Methods for directed differentiation of pluripotent stem cells to immune cells

ABSTRACT

Provided herein are methods for the efficient in vitro differentiation of somatic cell-derived pluripotent stem cells to hematopoietic precursor cells, and the further differentiation of the hematopoietic precursor cells into immune cells of various myeloid or lymphoid lineages, particularly T cells, NK cells, and dendritic cells. The pluripotent cells may be maintained and differentiated under defined conditions; thus, the use of mouse feeder cells or serum is not required in certain embodiments for the differentiation of the hematopoietic precursor cells.

The present application is a national phase application under 35 U.S.C.§ 371 of International Application No. PCT/US2016/057899, filed Oct. 20,2016, which claims the priority benefit of U.S. Provisional ApplicationsSer. No. 62/244,101, filed Oct. 20, 2015, and Ser. No. 62/404,470, filedOct. 5, 2016, the entire contents of each of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of molecularbiology. More particularly, it concerns methods and compositions for theproduction of immune cells from somatic cell-derived induced pluripotentstem cells (iPSCs).

2. Description of Related Art

Cell therapy methods have been developed in order to enhance the hostimmune response to tumors, viruses and bacterial pathogens. Cell therapymethods often involve the ex-vivo activation and expansion of T-cells.Examples of these types of treatments include the use of tumorinfiltrating lymphocyte (TIL) cells, cytotoxic T-cells, expanded tumordraining lymph node cells, and various other lymphocyte preparations.Due to the significant medical potential of hematopoietic stem andprogenitor cells, substantial work has been done to try to improvemethods for the differentiation of hematopoietic progenitor cells toimmune cells, such as T cells and NK cells.

In humans, induced pluripotent stem (iPS) cells are commonly generatedfrom dermal fibroblasts. However, the requirement for skin biopsies andthe need to expand fibroblast cells for several passages in vitro makeit a cumbersome source for generating patient-specific stem cells.Moreover, previous methods for reprogramming of human somatic cells areinconvenient because they need to obtain somatic cells directly from ahuman subject, or maintain the cells in a labor-intensive cell culturesystem. Therefore, there is a need to develop methods to inducepluripotent stem cells from alternative sources which are simple,convenient, and easily accessible. Accordingly, blood samples may besuch a source because blood may be collected from a patient or a healthyindividual, stored or transferred, for example, from a central unit fordistribution to one or more remote places. Thus, there currently existsa clear need for methods of reprogramming iPS cells from somatic cellsand then efficiently differentiating the somatic cell-derived iPS cellsto immune cells, such as T cells, NK cells, T/NK cells, and dendriticcells.

SUMMARY OF THE INVENTION

A first embodiment of the present disclosure provides a method ofproducing immune cells comprising obtaining pluripotent stem cells(PSCs), wherein the PSCs are reprogrammed from a population of somaticcells, differentiating the PSCs to hematopoietic precursor cells (HPCs),and culturing the HPCs under conditions to promote immune celldifferentiation, thereby producing immune cells.

In some aspects, the PSCs are induced pluripotent stem cells (iPSCs). Inother aspects, the PSCs are embryonic stem cells (ECSs). In someaspects, the PSCs are essentially free of integrated, exogenous viralelements.

In certain aspects, the immune cells are lymphoid cells. In particularaspects, the lymphoid cells are T cells, B cells, and/or NK cells. Insome aspects, the immune cells are myeloid cells. In specific aspects,the myeloid cells are dendritic cells.

In some aspects, the population of somatic cells is mammalian. Inparticular aspects, the population of somatic cells is human. In someaspects, the population of somatic cells are a population of blood cellsor a population of skin cells. In some aspects, the population of bloodcells has not been mobilized with extrinsically applied G-CSF. Incertain aspects, the population of blood cells comprises T cells, Bcells, and/or NK cells. In some aspects, the population of blood cellsis further defined as progenitor blood cells, peripheral bloodmononuclear cells, or lymphoblastoid cells. In certain aspects, thepopulation of blood cells is isolated from peripheral blood, umbilicalcord blood, or lymphoid tissue. In particular aspects, the lymphoidtissue comprises bone marrow, lymph node, or fetal liver. In specificaspects, the population of blood cells comprises T cells. In someaspects, the T cells are cultured in the presence of an anti-CD3antibody and/or an anti-CD28 antibody. In particular aspects, the Tcells are CD4⁺ or CD8⁺ T cells. In some aspects, the T cells are Thelper 1 (TH1) cells, T helper 2 (TH2) cells, TH17 cells, cytotoxic Tcells, regulatory T cells, natural killer T cells, naïve T cells, memoryT cells, or gamma delta T cells. In certain aspects, the reprogrammingcomprises introducing reprogramming factors into the population ofsomatic cells. In some aspects, reprogramming comprises introducing RNA,protein, or small molecules into the population of somatic cells.

In particular aspects, the population of somatic cells are blood cellsor skin cells. In some aspects, the reprogramming factors are encoded byone or more expression cassettes. In certain aspects, the reprogrammingfactors comprise two or more genes selected from the group consisting ofSox2, Oct4, cMyc, Klf4, Nanog, SV40 Large T antigen, and Lin28. In someaspects, the reprogramming factors comprise 3, 4, 5, or 6 of the genesselected from the group consisting of Sox2, Oct4, cMyc, Klf4, Nanog,SV40 Large T antigen, and Lin28. In certain aspects, the one or moreexpression cassettes are comprised in a reprogramming vector selectedfrom the group consisting of a viral vector, an episomal vector, and atransposon. In some aspects, the viral vector is further defined as aretroviral vector. In particular aspects, the episomal vector is furtherdefined as an Epstein-Barr virus (EBV)-based episomal vector. In someaspects, the reprogramming comprises culturing the cells under defined,feeder-free conditions.

In some aspects, the HPCs differentiate to at least 20 immune cells perHPC, such as at least 30, 40, 50, 60, 70, 70, 90, 100 or more immunecells per HSC. In some aspects, the PSCs differentiate to at least 20immune cells per PSC, such as at least 30, 40, 50, or more immune cellsper PSC.

In certain aspects, differentiating the PSCs to HPCs comprises thesequential steps of culturing or maintaining a plurality ofsubstantially undifferentiated pluripotent cells in a first definedmedia comprising at least one growth factor, incubating the cells in asecond defined media which is free or essentially free of IL-3, Flt3ligand, and GM-CSF, culturing the cells in a third defined mediacomprising BMP4, FGF2, and VEGF sufficient to expand or promotedifferentiation in a plurality of the cells, and culturing the cells ina fourth defined media comprising IL-3 and Flt3 ligand, sufficient toexpand or promote differentiation in a plurality of the cells. In someaspects, a plurality of the pluripotent cells are differentiated intoHPCs. In some aspects, the second defined media comprises blebbistatin.In certain aspects, the second defined media further comprises a GSK3inhibitor. In some aspects, the GSK3 inhibitor is CHIR99021. In someaspects, the second defined media further comprises BMP4, VEGF, andFGF2. In certain aspects, the cells are individualized prior toincubating the cells in the second defined media. In some aspects,incubating the cells in a second defined media, culturing the cells in athird defined media, and culturing the cells in a fourth defined mediais performed using amine culture plates. In certain aspects, the seconddefined media further comprises VEGF and FGF2. In particular aspects,the fourth defined media further comprises one or more of the cytokinesselected from the group consisting of IL-3, IL-6, SCF, TPO, and BMP4. Insome aspects, the fourth defined media comprises heparin. In someaspects, the method comprises culturing the cells at an atmosphericpressure of less than 25% oxygen, such as less than 24%, 23%, 22%, 21%,20%, 19%, 18%, 17%, 16%, 15%, 10%, or 5% oxygen. In some aspects, aplurality of the pluripotent cells form embryoid bodies (EBs). Incertain aspects, the HPCs express CD34. In particular aspects, the HPCsexpress at least two markers from the group consisting of CD43, CD34,CD31, CD41, CD235 and CD45.

In some aspects, conditions to promote immune cell differentiation arefurther defined as conditions to promote lymphoid differentiation. Incertain aspects, HPCs that express CD34 and CD43 are cultured underconditions to promote lymphoid differentiation. In some aspects,culturing the cells to promote lymphoid differentiation comprisesculturing HPCs in defined media on a surface coated with matrix and aNotch ligand, wherein the HPCs express one or more of the cell surfacemarkers selected from the group consisting of CD34, CD43, CD7, DLL4,CD144, and CD235, and maintaining the culture in the presence of one ormore cytokines, thereby producing lymphoid cells. In some aspects, theHPCs express CD144, CD34, CD45, and CD7. In particular aspects, the HPCsexpress CD144, CD34, CD45, and CD7.

In additional aspects, the first step for lymphoid differentiationfurther comprises isolating the HPCs that express one or more cellsurface markers. In some aspects, isolating comprises magnetic-activatedcell sorting (MACS). In certain aspects, the cells are cultured at anatmospheric pressure of less than 5% oxygen. In certain aspects, thecells are cultured at an atmospheric pressure of about 5% oxygen. Insome aspects, the defined media comprises ascorbic acid and/ornicotinamide. In certain aspects, the ascorbic acid is present at aconcentration of 50 μM to 1 mM, such as 90 μM to 100 μM. In someaspects, the nicotinamide is present at a concentration of 0.1 mM to 5mM. In some aspects, the nicotinamide is nicotinic acid. In someaspects, the matrix is extracellular matrix protein. In some aspects,the matrix is retronectin, collagen, laminin or fibronectin. Inparticular aspects, the matrix is retronectin. In some aspects, theNotch ligand is DLL4. In certain aspects, the DLL4 is DLL4:Fc chimeraprotein. In particular aspects, the one or more cytokines are selectedfrom the group consisting of SCF, TPO, IL-7, and Flt-3. In some aspects,the second step is one to six weeks, such as two to four weeks. In someaspects, the lymphoid cells express one or more of the markers selectedfrom the group consisting of CD8, CD7, CD45, CD5, CD4 and CD3. In someaspects, more than 5% of the lymphoid cells are positive for at leasttwo of the markers. In particular aspects, more than 10% of the lymphoidcells are positive for at least two of the markers.

In some aspects, conditions to promote immune cell differentiation arefurther defined as conditions to promote myeloid differentiation. Incertain aspects, culturing the HPCs under conditions to promote myeloiddifferentiation comprises culturing the HPCs in a first defined mediacomprising TPO, SCF, and Flt3 ligand, thereby producing a population ofmyeloid cells, and incubating the cells in a second defined mediaessentially free of TPO, SCF, and Flt3 ligand, thereby producing anenriched population of myeloid cells. In some aspects, the first definedmedia further comprises IL-6 and IL-3. In certain aspects, the seconddefined media comprises GM-CSF. In particular aspects, at least 50% ofthe population of myeloid cells produced in the first step are positivefor CD45, CD43, and CD31. In some aspects, the population of myeloidcells positive for CD45, CD43, and CD31 has essentially no expression ofCD34. In particular aspects, at least 80% of the enriched population ofmyeloid cells is CD43⁺, CD45⁺, CD31⁺, and CD34⁻. In some aspects, thesecond step is for 5 to 10 days.

In certain aspects, the method further comprises differentiating theenriched population of myeloid cells to dendritic cells. In someaspects, differentiating the enriched population of myeloid cells todendritic cells comprises culturing the enriched population of myeloidcells in a defined media comprising GM-CSF, IL-4, and TNFα, therebyproducing dendritic cells. In some aspects, the defined media furthercomprises lipoproteins. In certain aspects, the dendritic cells expressone or more of the markers selected from the group consisting of CD209,CD1a, HLA-DR, CD11c, CD14, CD83, and CD86. In particular aspects, thedendritic cells have essentially no expression of CD12.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1D: (A) Schematic of feeder free method to derive T, NK, andNK/T cells from iPSCs. The iPSCs are differentiated to HPCs withlymphoid and myeloid potential by the 3D process and the Day 7-10progenitors are then subjected to a 2D differentiation process togenerate T, NK, and NK/T cells (B) Schematic of feeder free method toderive dendritic cells from iPSCs. The iPSCs are differentiated to HPCswith lymphoid and myeloid potential by the 3D process and the Day 12progenitors are further differentiated to generate a myeloid progenitorand subjected to a step wise 3D differentiation process to generatedendritic cells (C) HPCs were generated from various blood cell-derivediPSC cell lines at day 12 of differentiation and the percentage ofCD43⁺/CD34⁺ cells quantified by flow cytometry is shown. (D) Theefficiency of HPCs generation from Day 12 iPSCs is shown as the ratio ofthe absolute number of HPCs generated per input number of iPSCs.

FIG. 2: Flow cytometry scatter plots are shown which identify lymphoidand lympho-myeloid populations during lymphoid differentiation of theDay 7-10 HPCs. The presence of pre T, NK, and NK/T cells was determined.

FIGS. 3A-3D: (A) Representative staining profile to detect the emergenceof lymphoid cells from virally and episomally reprogrammed iPSCs,including cord blood-derived iPSCs, virally reprogrammed T cell iPSCs(TiPSCs), and episomally reprogrammed progenitor blood cell iPSCs(01279.107.3908). The cells were stained for the surface 2The percentageof cells were quantified by flow cytometry under FSC-SSC and thelymphoid scatter gates. (EB7=Day 7, EB8=Day 8, EB9=Day 9, EB10=Day 10,and EB11=Day 11. (B) Day 7-11 HPCs differentiated from TiPSCs (top:Total HPCs; bottom: CD43⁺CD34⁺ HPCs) were further differentiated tolymphoid cells and stained for the surface expression of CD45, CD7, andCD5. The percentage of cells were quantified by flow cytometry underFSC-SSC and the lymphoid scatter gates. (C) Day 7-11 HPCs generated fromTiPSCs (top: Total HPCs; bottom: CD43⁺CD34⁺ HPCs) were differentiatedunder hypoxic conditions and stained for the surface expression of CD56,CD8, and CD3. (D) The efficiency of generating lymphoid cells fromTiPSCs is shown (left: Total HPCs; right: CD43⁺CD34⁺ HPCs).

FIG. 4: Day 7-11 HPCs differentiated from TiPSCs (e.g., virallyreprogrammed TiPSCs1E or episomally reprogrammed 01279.107.3902) wereanalyzed for the expression of CD43/CD34, CD34, D114, D114/CD144,CD31/CD144, and CD235 at various days of HPC differentiation from day 7to day 11. The percentage of cells positive for each set of markers ifshown. The CD43/CD34 expression is shown in the left column, CD34 rightcolumn, DLL4/CD144 bottom line, CD31/CD144 middle line, and CD235 topline.

FIG. 5: Schematic of magnetic sorting strategy to detect lymphoidprogenitors during HPC differentiation. The markers of interest includedCD31, CD34, CD144, CD43, CD45, CD7, CD235 FLK1 (also known as KDR,VEGFR2, CD309) and DLL4. At Day 8 of differentiation, the cells weresorted into various fractions based upon the markers of interest andthen subjected to the lymphoid differentiation process.

FIGS. 6A-6B: (A) The percentage of CD3 positive cells in each positiveand negative fraction of cells from the magnetic sorting strategy ofFIG. 5 is shown with unsorted cells as the control. (B) The foldenrichment of T cells generated from the HPCs is shown for the positiveand negative fractions from the magnetic sorting strategy of FIG. 5 withunsorted cells as the control.

FIGS. 7A-7B: (A) The percentage of CD3 positive cells for each positivefraction of cells from the magnetic sorting of FIG. 5 after 4 weeks oflymphoid differentiation from TiPSCs is shown. (B) FACS plot of 5 weekdifferentiation from TiPSCs1E cells. Flow cytometry analysis of the CD3positive cells for expression of emerging lymphoid cells are CD7+, CD5+,CD3+, CD8+, CD56+CD335+, CD161+, TCR αβ+, TCR γδ−,

FIGS. 8A-8B: (A) Efficiency of TiPCs lymphoid differentiation processfrom HPCs at day 16 of both positive and negative magnetic sortedfractions is shown as the ratio of input HPCs to output lymphoid cells.(B) Cumulative efficiency of the differentiation process at the end of 4weeks starting with the positive magnetic sorting fractions.

FIGS. 9A-9B: (A) Phenotypic analysis of dendritic cells derived fromiPSC 02179 (MeCP2 WT) on day 42 of differentiation. The cells werestained for the cell surface expression of myeloid dendritic markersCD205, CD209, HLA-DR, CD1a, CD1c, CD80, CD11c, CD80, CD86 and CD83 (B)Representative FACS plot histograms for quantifying the expression ofvarious cell surface markers expressed on dendritic cells.

FIG. 10: DQ TM ovalbumin (DQ-OVA, Invitrogen) was dissolved at 1 mg/mlin PBS and added to iPSC derived DCs at 100 ug/ml. The cells wereincubated either at 37° C. or at 4° C., washed twice with FACS bufferand analyzed on the Accuri flow cytometer. The specific uptake of OVAwas demonstrated by iPSC derived DCs at 37° C. compared to thenon-specific OVA uptake at 4° C.

FIG. 11: Diagram representing feeder-free and serum-free T and NK celldifferentiation of hPCS. PSCs were first differentiated to CD34+hematopoietic progenitor cells (HPC) in suspension (3D) embryonic body(EB) culture through successive steps of aggregate formation, mesoderminduction and HPC differentiation during 9 days. CD34+ cells wereisolated by MACS using direct CD34 paramagnetic beads (Miltenyi Biotec)and transferred to DLL4+ retronectin coated plates for T/NKdifferentiation during 2 weeks. T cells could further be expanded during2 weeks in culture on anti-CD3 mAb (OKT3)+ retronectin coated (both at0.5 μg/cm²) plates in T-EM (ImmunoCult-XF T cell expansion medium (StemCell Technologies)) supplemented with IL2 alone or in combination withother T cell growth promoting cytokines (IL7, IL15, IL21).Abbreviations: SFDM, serum-free differentiation medium; TCDM, T celldifferentiation medium; TCEM, T cell expansion medium; MACS,magnet-activated cell sorting.

FIG. 12: Flow cytometric analysis of T/NK differentiation cultures. PSC(1C TiPSC)-derived CD34+ cells after 2 weeks in T/NK differentiationconditions develop a typical lymphoid cell population defined by lowFSC/SSC parameters (left dot-plot). This lymphoid population containsmostly CD3+T and CD56+CD3− NK cells (middle dot-plot). T cell populationincludes CD4+ and CD8+ single and double positive cells as well assignificant proportion of double negative cells (right dot plot).

FIG. 13: The yield of different cell populations throughoutdifferentiation. The yields of each respective cell type are expressedas a ratio of output to input absolute cell numbers at each stage ofcell derivation depicted in the diagram. For example, 1.5 CD34+ cellyield indicates that in average 1.5 (output) CD34+ cells can be derivedfrom 1 (input) PSC cell. Accordingly, 102 T cell yield indicates that102 (output) T cells can be derived from 1 (input) CD34+ cells.

FIG. 14: Phenotype of PSC-derived T cells. PSC-derived T cells (CD3+)differentiated and expanded during 4 weeks express α/β TCR (not γ/δ orinvariant Vα24 NKT TCR) and typical T cell markers CD5, CD27, CD7. Theyalso express NK associated (CD161, CD94) and activation (CD69) markers.

FIG. 15: Expansion of PSC-derived T cells. Immobilized anti-CD3antibodies (iCD3) are minimally required and sufficient to achieveexpansion of PSC-derived T cells (bar graph). Soluble stimulating CD3and CD28 mAb (sCD3, sCD28) were not effective either alone (not shown)or in combination (sCD3+sCD28), or when added to iCD3 (iCD3+sCD28). Tcells proliferating in the expansion cultures acquire a characteristicmorphology of irregularly shaped lymphoblasts (photograph). In contrastto relatively heterogeneous input cell population, cells harvested from2 week T cell expansion are essentially pure CD3+ T cells, which alsoexpress CD56 and acquire CD8 expression (flow cytometry dot plots).

FIGS. 16A-16H: (A) Representative photograph of day 8 HPC cultures: TheHPCs bud off from the underlying endothelial layer. (B) Schematicrepresentation of the 2D HPC differentiation process. (C) Generation ofHPCs from 01279.107.3902 (MeCP2 KO) cell line at days 7-10 ofdifferentiation. (D) Generation of HPCs from TiPSCs1E cell line at days7-10 of differentiation. The cells were harvested and the percentage ofCD43/CD34 cells was quantified by flow cytometry. (E) Efficiency ofgeneration of HPCs from iPSCs. The efficiency of the process iscalculated by dividing the absolute number of HPCs generated per inputnumber of iPSCs. (F) Analysis of Pre T and Pre NK cells. HPCs generatedfrom iPSC 0.1279.107.3902 (MeCP2 KO) were harvested on days 7-10 andcryopreserved. HPCs were thawed and plated at a density of 25K/cm² onRetronectin-DLL4 coated plates. The cells were placed in Serum FreeDefined (SFD) media containing 1% Glutamax, 1% Penicillin Streptomycin,95 μM L-ascorbic acid 2-phosphate, 50 ng/mL stem cell factor (SCF),thrombopoietin (TPO), Flt-3 Ligand (Flt-3), and IL-7 to stimulatelymphoid differentiation under hypoxic and conditions. The cells werefed with fresh media every 48 hrs and harvested on day 14 using coldPBS. The cells were stained for the surface expression of CD4, CD7, CD5,CD56, CD8, and CD3. The percentage of cells were quantified by flowcytometry under lymphoid scatter gate. The presence of T, NK and NK/Tcells were quantified. (G) Analysis of Pre T and Pre NK cells. The cellswere stained for the surface expression of CD4, CD7, CD5, CD56, CD8, andCD3. The percentage of cells were quantified by flow cytometry underlymphoid scatter gate. (H) Efficiency of generation of T cells fromHPCs. The efficiency of the process is calculated by dividing theabsolute number of T cells (CD3+) generated per input number of HPCs(CD43/34+).

FIGS. 17A-17C: (A) Schematic representation of 3D HPC differentiationprocess using iPSCs adapted to feeder free growth on Matrigel orVitronectin in the presence of E8 media and hypoxic conditions. Thefirst stage of HPC differentiation is driven by BMP4, VEGF and FGF for 4days and the second stage of differentiation is driven by placing cellsin media containing Heparin, SCF, TPO, Flt-3 Ligand, IL-3 and IL-6. (B)Engineering strategy to generate a MeCP2 KO in male iPSC cell line 2.038to create 9006 (01279.107.003902). (C) Depiction of the amino acidalignment of MeCP2 variants 001, 002, 005 and 008 derived from 01279iPSCs transfected with MeCP2 TALENS and Donor plasmid p1553. Allvariants (001, 002, 005 and 008) do not code for a MethylCpG bindingdomain.

FIGS. 18A-18C: Quantification of Pre T and Pre NK cells iPSC Tips 1Eharvested on day 7-11 of HPC differentiation and placed on Ret-DLL4coated plates to initiate lymphoid differentiation at a density of2.5×10⁴/cm². The percentages of CD8+CD3+(T cells), CD56+/CD8+(NK cells),and CD56+/CD3+(NK/T cells) in 01279 (MeCP2WT) cells maintained underhypoxic conditions (A) and normoxic conditions (B) as well as01279.107.3902 cells (MeCP2K0) (C) was determined. (A-B) TheNK cellshave the highest percentage of positive cells, followed by NK/T cells,and T cells. (C) From Day 7 to Day 10, the percentage of positive cellsfrom top to bottom are T cells, NK/T cells, and NK cells. At Day 11, thehighest percentage of positive cells are NK cells followed by NK/T cellsand T cells.

FIGS. 19A-19C: Gating strategy for identifying lymphoid cells generatedin vitro. (A) General scatter profile of lymphocytes from adult humanperipheral blood. (B) Scatter profile of lymphoid cells at day 18 ofdifferentiation. The FSC-SSC gate and the lymphoid gate are illustrated.(C) Gating live cells within the FSC-SSC scatter and lymphoid scatterusing propidium iodide.

FIG. 20: Gating strategy for identifying lymphoid cells generated invitro. A scatter profile of lymphoid cells at day 18 of differentiationis shown. The FSC-SSC gate and the lymphoid gate are illustrated. Livecells were gated within the FSC-SSC scatter and lymphoid scatter usingpropidium iodide followed by staining for CD7 and CD5 positive cells byflow cytometry.

FIGS. 21A-21B: Quantification of NK (CD3−/CD56+) cells on day 7-11 ofHPC of differentiation and placed on Ret-DLL4 coated plates to initiatelymphoid differentiation at a density of 2.5×10⁴/cm². The percentages ofdouble positive, CD56+/CD3− under the all live FSC-SSC gate (A) andlymphoid gate (B) was determined for iPSC clones containing MeCp2 WT andMeCp2KO status.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present disclosure overcomes problems associated with currenttechnologies by providing highly efficient methods for generating immunecells from induced pluripotent stem cells which have been reprogrammedfrom a starting population of somatic cells (e.g., blood cells). Theimmune cells produced by the current methods can include T cells, NKcells, T/NK cells, and dendritic cells. In some aspects, the startingpopulation of somatic cells comprises T cells. The T cells may beisolated from various sources, such as a blood sample.

The population of somatic cells is reprogrammed to iPSCs by theintroduction of reprogramming factors, such as through a viral orepisomal vector. These somatic-cell derived iPSCs (e.g., T-cell derivediPSCs (TiPSCs)) are then differentiated to hematopoietic precursorcells. In one method, the differentiation process involves WNTactivation, culture with hematopoetic inductive cytokines and CD34⁺ cellisolation. Finally, the HPCs are then differentiated to immune cells,such as lymphoid cells (e.g., T, NK, and T/NK cells) and myeloid cells(e.g., dendritic cells).

The differentiation to lymphoid cells may be through the use ofRetroNectin and DLL-4 as a feeder free matrix. The T celldifferentiation may be further enhanced by the use of ascorbic acid toincrease the efficiency and maturation as well as by culturing underhypoxic conditions. Interestingly, the inventors have determined anoptimal timeframe during HPC differentiation (e.g., day 7-11) forlymphoid potential. These HPCs with lymphoid potential may be identifiedby expression of CD34 and CD43. In addition, HPCs with enhanced lymphoidpotential may be isolated by sorting for fractions of cells positive fortwo or more of the markers CD144, CD34, CD45, and CD7. In some aspects,the progenitor for derivation of dendritic cells is a common myeloidprogenitor that emerges at around day 16 of HPC differentiation.

The lymphoid cells produced from the somatic cell-derived PSCs caninclude T cell, NK cells and T/NK cells which retain theircharacteristic T-cell receptor (TCR) gene rearrangements, a propertywhich could be exploited, for example, as a genetic tracking marker orin re-differentiation experiments to study human T-cell development. Aparticular advantage of the present disclosure lies in rearranged andreduced V, D, J gene segments of T-cell receptors which may be retainedin the differentiated T cells. This serves as a specific characteristicor “barcode” of different clonal populations of T cell-derived iPScells, and also help differentiates those iPS cells from pluripotentstem cells which have not undergone V(D)J recombination.

Thus, the methods of the present disclosure could provide unlimitednumbers of immune cells, such as T cells, NK cells, T/NK cells, anddendritic cells, for a wide range of applications such as stabletransplantation in vivo, screening of compounds in vitro, andelucidating the mechanisms of hematological diseases and injuries.

I. Definitions

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis therefore well below 0.05%, preferably below 0.01%. Most preferred isa composition in which no amount of the specified component can bedetected with standard analytical methods.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The term “exogenous,” when used in relation to a protein, gene, nucleicacid, or polynucleotide in a cell or organism refers to a protein, gene,nucleic acid, or polynucleotide that has been introduced into the cellor organism by artificial or natural means; or in relation to a cell,the term refers to a cell that was isolated and subsequently introducedto other cells or to an organism by artificial or natural means. Anexogenous nucleic acid may be from a different organism or cell, or itmay be one or more additional copies of a nucleic acid that occursnaturally within the organism or cell. An exogenous cell may be from adifferent organism, or it may be from the same organism. By way of anon-limiting example, an exogenous nucleic acid is one that is in achromosomal location different from where it would be in natural cells,or is otherwise flanked by a different nucleic acid sequence than thatfound in nature.

By “expression construct” or “expression cassette” is meant a nucleicacid molecule that is capable of directing transcription. An expressionconstruct includes, at a minimum, one or more transcriptional controlelements (such as promoters, enhancers or a structure functionallyequivalent thereof) that direct gene expression in one or more desiredcell types, tissues or organs. Additional elements, such as atranscription termination signal, may also be included.

A “vector” or “construct” (sometimes referred to as a gene deliverysystem or gene transfer “vehicle”) refers to a macromolecule or complexof molecules comprising a polynucleotide to be delivered to a host cell,either in vitro or in vivo.

A “plasmid,” a common type of a vector, is an extra-chromosomal DNAmolecule separate from the chromosomal DNA that is capable ofreplicating independently of the chromosomal DNA. In certain cases, itis circular and double-stranded.

An “origin of replication” (“ori”) or “replication origin” is a DNAsequence, e.g., in a lymphotrophic herpes virus, that when present in aplasmid in a cell is capable of maintaining linked sequences in theplasmid and/or a site at or near where DNA synthesis initiates. As anexample, an ori for EBV (Ebstein-Barr virus) includes FR sequences (20imperfect copies of a 30 bp repeat), and preferably DS sequences;however, other sites in EBV bind EBNA-1, e.g., Rep* sequences cansubstitute for DS as an origin of replication (Kirshmaier and Sugden,1998). Thus, a replication origin of EBV includes FR, DS or Rep*sequences or any functionally equivalent sequences through nucleic acidmodifications or synthetic combination derived therefrom. For example,the present disclosure may also use genetically engineered replicationorigin of EBV, such as by insertion or mutation of individual elements,as specifically described in Lindner et al., 2008.

A “gene,” “polynucleotide,” “coding region,” “sequence,” “segment,”“fragment,” or “transgene” that “encodes” a particular protein, is anucleic acid molecule that is transcribed and optionally also translatedinto a gene product, e.g., a polypeptide, in vitro or in vivo whenplaced under the control of appropriate regulatory sequences. The codingregion may be present in either a cDNA, genomic DNA, or RNA form. Whenpresent in a DNA form, the nucleic acid molecule may be single-stranded(i.e., the sense strand) or double-stranded. The boundaries of a codingregion are determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A gene can include,but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomicDNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNAsequences. A transcription termination sequence will usually be located3′ to the gene sequence.

The term “control elements” refers collectively to promoter regions,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (IRES), enhancers, splice junctions, and the like, whichcollectively provide for the replication, transcription,post-transcriptional processing, and translation of a coding sequence ina recipient cell. Not all of these control elements need be present solong as the selected coding sequence is capable of being replicated,transcribed, and translated in an appropriate host cell.

The term “promoter” is used herein in its ordinary sense to refer to anucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene that is capable of bindingRNA polymerase and initiating transcription of a downstream (3′direction) coding sequence. It may contain genetic elements at whichregulatory proteins and molecules may bind, such as RNA polymerase andother transcription factors, to initiate the specific transcription of anucleic acid sequence. The phrases “operatively positioned,”“operatively linked,” “under control,” and “under transcriptionalcontrol” mean that a promoter is in a correct functional location and/ororientation in relation to a nucleic acid sequence to controltranscriptional initiation and/or expression of that sequence.

By “enhancer” is meant a nucleic acid sequence that, when positionedproximate to a promoter, confers increased transcription activityrelative to the transcription activity resulting from the promoter inthe absence of the enhancer domain.

By “operably linked” or co-expressed” with reference to nucleic acidmolecules is meant that two or more nucleic acid molecules (e.g., anucleic acid molecule to be transcribed, a promoter, and an enhancerelement) are connected in such a way as to permit transcription of thenucleic acid molecule. “Operably linked” or “co-expressed” withreference to peptide and/or polypeptide molecules means that two or morepeptide and/or polypeptide molecules are connected in such a way as toyield a single polypeptide chain, i.e., a fusion polypeptide, having atleast one property of each peptide and/or polypeptide component of thefusion. The fusion polypeptide is preferably chimeric, i.e., composed ofheterologous molecules.

“Homology” refers to the percent of identity between two polynucleotidesor two polypeptides. The correspondence between one sequence and anothercan be determined by techniques known in the art. For example, homologycan be determined by a direct comparison of the sequence informationbetween two polypeptide molecules by aligning the sequence informationand using readily available computer programs. Alternatively, homologycan be determined by hybridization of polynucleotides under conditionsthat promote the formation of stable duplexes between homologousregions, followed by digestion with single strand-specific nuclease(s),and size determination of the digested fragments. Two DNA, or twopolypeptide, sequences are “substantially homologous” to each other whenat least about 80%, preferably at least about 90%, and most preferablyat least about 95% of the nucleotides, or amino acids, respectivelymatch over a defined length of the molecules, as determined using themethods above.

The term “cell” is herein used in its broadest sense in the art andrefers to a living body that is a structural unit of tissue of amulticellular organism, is surrounded by a membrane structure thatisolates it from the outside, has the capability of self-replicating,and has genetic information and a mechanism for expressing it. Cellsused herein may be naturally-occurring cells or artificially modifiedcells (e.g., fusion cells, genetically modified cells, etc.).

The term “stem cell” refers herein to a cell that under suitableconditions is capable of differentiating into a diverse range ofspecialized cell types, while under other suitable conditions is capableof self-renewing and remaining in an essentially undifferentiatedpluripotent state. The term “stem cell” also encompasses a pluripotentcell, multipotent cell, precursor cell and progenitor cell. Exemplaryhuman stem cells can be obtained from hematopoietic or mesenchymal stemcells obtained from bone marrow tissue, embryonic stem cells obtainedfrom embryonic tissue, or embryonic germ cells obtained from genitaltissue of a fetus. Exemplary pluripotent stem cells can also be producedfrom somatic cells by reprogramming them to a pluripotent state by theexpression of certain transcription factors associated withpluripotency; these cells are called “induced pluripotent stem cells” or“iPSCs or iPS cells”.

An “embryonic stem (ES) cell” is an undifferentiated pluripotent cellwhich is obtained from an embryo in an early stage, such as the innercell mass at the blastocyst stage, or produced by artificial means (e.g.nuclear transfer) and can give rise to any differentiated cell type inan embryo or an adult, including germ cells (e.g. sperm and eggs).

“Induced pluripotent stem cells (iPSCs or iPS cells)” are cellsgenerated by reprogramming a somatic cell by expressing or inducingexpression of a combination of factors (herein referred to asreprogramming factors). iPS cells can be generated using fetal,postnatal, newborn, juvenile, or adult somatic cells. In certainembodiments, factors that can be used to reprogram somatic cells topluripotent stem cells include, for example, Oct4 (sometimes referred toas Oct 3/4), Sox2, c-Myc, Klf4, Nanog, and Lin28. In some embodiments,somatic cells are reprogrammed by expressing at least two reprogrammingfactors, at least three reprogramming factors, at least fourreprogramming factors, at least five reprogramming factors, at least sixreprogramming factors, or at least seven reprogramming factors toreprogram a somatic cell to a pluripotent stem cell.

“Hematopoietic progenitor cells” or “hematopoietic precursor cells”refers to cells which are committed to a hematopoietic lineage but arecapable of further hematopoietic differentiation and includehematopoietic stem cells, multipotential hematopoietic stem cells,common myeloid progenitors, megakaryocyte progenitors, erythrocyteprogenitors, and lymphoid progenitors. Hematopoietic stem cells (HSCs)are multipotent stem cells that give rise to all the blood cell typesincluding myeloid (monocytes and macrophages, granulocytes (neutrophils,basophils, eosinophils, and mast cells), erythrocytes,megakaryocytes/platelets, dendritic cells), and lymphoid lineages(T-cells, B-cells, NK-cells) (see e.g., Doulatov et al., 2012; Notta etal., 2015). A “multilymphoid progenitor” (MLP) is defined to describeany progenitor that gives rise to all lymphoid lineages (B, T, and NKcells), but that may or may not have other (myeloid) potentials(Doulatov et al., 2010) and is CD45RA⁺/CD10⁺/CD7⁻. Any B, T, and NKprogenitor can be referred to as an MLP. A “common myeloid progenitor”(CMP) refers to a common myeloid progenitor is defined by the expressionof CD45+/CD31+/CD43+/CD34⁻ cells that can give rise to granulocytes,monocytes, megakaryocytes and erythrocytes. The hematopoietic progenitorcells may express CD34. The hematopoietic progenitor cells mayco-express CD133 and be negative for CD38 expression. Hematopoieticprecursor cells include CD34⁺/CD45⁺ hematopoietic precursor cells andCD34⁺/CD45⁺/CD43⁺ hematopoietic precursor cells. The CD34⁺/CD43⁺/CD45+hematopoietic precursor cells may be highly enriched for myeloidprogenitors. Hematopoietic cells also include various subsets ofprimitive hematopoietic cells including: CD34⁻/CD133⁺/CD38⁻ (primitivehematopoietic precursor cells), CD43(+)CD235a(+)CD41a(+/−)(erythro-megakaryopoietic), lin(−)CD34(+)CD43(+)CD45(−) (multipotent),and lin(−) CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells, CD133+/ALDH+(aldehydehehydrogenase) (e.g., Hess et al. 2004; Christ et al., 2007).It is anticipated that any of these primitive hematopoietic cell typesor hematopoietic precursor cells may be converted into iPS cells asdescribed herein. In some aspects, the cells may include Mast cells,Langerhan's cells, Osteoclasts, NK cells, T cells, CIK T cells, or othersubtypes of T cells, NK cells, and B cells.

As used herein, the term “immune cell(s)” refers to cells of the immunesystem, including, but not limited to, T cells, NK cells, T/NK cells,dendritic cells, macrophages, B cells, neutrophils, erythrocytes,monocytes, basophils, neutrophils, mast cells, eosinphils, and anycombination thereof.

An “activator” of a T cell or a condition that will activate a T cellrefers to a stimulus that activates T cells and include antigens, whichmay be presented on antigen presenting cells or on other surfaces;polyclonal activators, which bind to many T cell receptor (TCR)complexes regardless of specificity, and include lectins, e.g.,concanavalin-A (Con-A) and phytohemagglutinin (PHA) and agents such asantibodies that bind specifically to invariant framework epitopes on TCRor CD3 proteins; and superantigens, which stimulate a significant numberof T cells, and include, e.g., enterotoxins, such as Staphyloccalenterotoxins.

The terms “T lymphocyte” and “T cell” are used interchangeably, andrefer to a cell that expresses a T cell antigen receptor (TCR) capableof recognizing antigen when displayed on the surface of antigenpresenting cells or matrix together with one or more MHC molecules or,one or more non-classical MHC molecules.

The term “T cell” refers to T lymphocytes as defined in the art and isintended to include thymocytes, immature T lymphocytes, mature Tlymphocytes, resting T lymphocytes, or activated T lymphocytes. The Tcells can be CD4⁺ T cells, CD8⁺ T cells, CD4⁺CD8⁺ T cells, or CD4⁻CD8⁻cells. The T cells can also be T helper cells, such as T helper 1 (TH1),or T helper 2 (TH2) cells, or TH17 cells, as well as cytotoxic T cells,regulatory T cells, natural killer T cells, naïve T cells, memory Tcells, or gamma delta T cells (Wilson et al., 2009; Wynn, 2005; Ladi etal., 2006). T cells that differ from each other by at least one marker,such as CD4, are referred to herein as “subsets” of T cells.

“CD4⁺ T cells” refers to a subset of T cells that express CD4 on theirsurface and are associated with cell-mediated immune response. They arecharacterized by the secretion profiles following stimulation, which mayinclude secretion of cytokines such as IFN-gamma, TNF-alpha, IL-2, IL-4and IL-10. “CD4” are 55-kD glycoproteins originally defined asdifferentiation antigens on T-lymphocytes, but also found on other cellsincluding monocytes/macrophages. CD4 antigens are members of theimmunoglobulin supergene family and are implicated as associativerecognition elements in MHC (major histocompatibility complex) classII-restricted immune responses. On T-lymphocytes they define thehelper/inducer subset.

“CD8⁺ T cells” refers to a subset of T cells which express CD8 on theirsurface, are MHC class I-restricted, and function as cytotoxic T cells.“CD8” molecules are differentiation antigens found on thymocytes and oncytotoxic and suppressor T-lymphocytes. CD8 antigens are members of theimmunoglobulin supergene family and are associative recognition elementsin major histocompatibility complex class I-restricted interactions.

“Pluripotent stem cell” refers to a stem cell that has the potential todifferentiate into all cells constituting one or more tissues or organs,or preferably, any of the three germ layers: endoderm (interior stomachlining, gastrointestinal tract, the lungs), mesoderm (muscle, bone,blood, urogenital), or ectoderm (epidermal tissues and nervous system).

As used herein, the term “somatic cell” refers to any cell other thangerm cells, such as an egg, a sperm, or the like, which does notdirectly transfer its DNA to the next generation. Typically, somaticcells have limited or no pluripotency. Somatic cells used herein may benaturally-occurring or genetically modified.

“Programming” is a process that alters the type of progeny a cell canproduce. For example, a cell has been programmed when it has beenaltered so that it can form progeny of at least one new cell type,either in culture or in vivo, as compared to what it would have beenable to form under the same conditions without programming. This meansthat after sufficient proliferation, a measurable proportion of progenyhaving phenotypic characteristics of the new cell type are observed, ifessentially no such progeny could form before programming;alternatively, the proportion having characteristics of the new celltype is measurably more than before programming. This process includesdifferentiation, dedifferentiation and transdifferentiation.

“Differentiation” is the process by which a less specialized cellbecomes a more specialized cell type. “Dedifferentiation” is a cellularprocess in which a partially or terminally differentiated cell revertsto an earlier developmental stage, such as pluripotency or multipotency.“Transdifferentiation” is a process of transforming one differentiatedcell type into another differentiated cell type. Typically,transdifferentiation by programming occurs without the cells passingthrough an intermediate pluripotency stage—i.e., the cells areprogrammed directly from one differentiated cell type to anotherdifferentiated cell type. Under certain conditions, the proportion ofprogeny with characteristics of the new cell type may be at least about1%, 5%, 25% or more in order of increasing preference.

“Reprogramming” is a process that confers on a cell a measurablyincreased capacity to form progeny of at least one new cell type, eitherin culture or in vivo, than it would have under the same conditionswithout reprogramming. More specifically, reprogramming is a processthat confers on a somatic cell a pluripotent potential. This means thatafter sufficient proliferation, a measurable proportion of progenyhaving phenotypic characteristics of the new cell type if essentially nosuch progeny could form before reprogramming; otherwise, the proportionhaving characteristics of the new cell type is measurably more thanbefore reprogramming. Under certain conditions, the proportion ofprogeny with characteristics of the new cell type may be at least about1%, 5%, 25% or more in order of increasing preference.

The term “forward programming” refers to the programming of amultipotent or pluripotent cell, as opposed to a differentiated somaticcell that has no pluripotency, by the provision of one or more specificlineage-determining genes or gene products to the multipotent orpluripotent cell. For example, forward programming may describe theprocess of programming ESCs or iPSCs to hematopoietic precursor cells orother precursor cells, or to hematopoietic cells or other differentiatedsomatic cells.

As used herein, the term “subject” or “subject in need thereof” refersto a mammal, preferably a human being, male or female at any age that isin need of a cell or tissue transplantation. Typically the subject is inneed of cell or tissue transplantation (also referred to herein asrecipient) due to a disorder or a pathological or undesired condition,state, or syndrome, or a physical, morphological or physiologicalabnormality which is amenable to treatment via cell or tissuetransplantation.

As used herein, a “disruption” of a gene refers to the elimination orreduction of expression of one or more gene products encoded by thesubject gene in a cell, compared to the level of expression of the geneproduct in the absence of the disruption. Exemplary gene productsinclude mRNA and protein products encoded by the gene. Disruption insome cases is transient or reversible and in other cases is permanent.Disruption in some cases is of a functional or full length protein ormRNA, despite the fact that a truncated or non-functional product may beproduced. In some embodiments herein, gene activity or function, asopposed to expression, is disrupted. Gene disruption is generallyinduced by artificial methods, i.e., by addition or introduction of acompound, molecule, complex, or composition, and/or by disruption ofnucleic acid of or associated with the gene, such as at the DNA level.Exemplary methods for gene disruption include gene silencing, knockdown,knockout, and/or gene disruption techniques, such as gene editing.Examples include antisense technology, such as RNAi, siRNA, shRNA,and/or ribozymes, which generally result in transient reduction ofexpression, as well as gene editing techniques which result in targetedgene inactivation or disruption, e.g., by induction of breaks and/orhomologous recombination. Examples include insertions, mutations, anddeletions. The disruptions typically result in the repression and/orcomplete absence of expression of a normal or “wild type” productencoded by the gene. Exemplary of such gene disruptions are insertions,frameshift and missense mutations, deletions, knock-in, and knock-out ofthe gene or part of the gene, including deletions of the entire gene.Such disruptions can occur in the coding region, e.g., in one or moreexons, resulting in the inability to produce a full-length product,functional product, or any product, such as by insertion of a stopcodon. Such disruptions may also occur by disruptions in the promoter orenhancer or other region affecting activation of transcription, so as toprevent transcription of the gene. Gene disruptions include genetargeting, including targeted gene inactivation by homologousrecombination.

“Notch ligand” is a protein capable of binding to a Notch receptorpolypeptide present in the membrane of a number of different mammaliancells such as hematopoietic stem cells. The Notch receptors that havebeen identified in human cells include Notch-1, Notch-2, Notch-3, andNotch-4. Notch ligands typically have a DSL domain (D-Delta, S-Serrate,and L-Lag2) comprising 20 to 22 amino acids at the amino terminus andbetween 3 to 8 EGF-like repeats (Furie and Furie, 1988; Knust et al.,1987; Suzuki et al., 1987) on the extracellular surface.

II. Somatic Cell-Derived iPSCs

A. Starting Population of Somatic Cells

Embodiments of the present disclosure concern a starting population ofsomatic cells (e.g., blood cells or skin cells) which are reprogrammedto iPSCs. The population of blood cells can include peripheral bloodmononuclear cells (PBMC), whole blood or fractions thereof containingmixed populations, spleen cells, bone marrow cells, tumor infiltratinglymphocytes, cells obtained by leukapheresis, biopsy tissue, and lymphnodes, e.g., lymph nodes draining from a tumor. Suitable donors includeimmunized donors, non-immunized (naive) donors, treated or untreateddonors. A “treated” donor is one that has been exposed to one or morebiological modifiers. An “untreated” donor has not been exposed to oneor more biological modifiers.

In some aspects, the population of blood cells comprises T cells. The Tcells can be a purified population of T cells, or alternatively the Tcells can be in a population with cells of a different type, such as Bcells and/or other peripheral blood cells. The T cells can be a purifiedpopulation of a subset of T cells, such as CD4⁺ T cells, or they can bea population of T cells comprising different subsets of T cells. Inanother embodiment, the T cells are T cell clones that have beenmaintained in culture for extended periods of time. T cell clones can betransformed to different degrees. In a specific embodiment, the T cellsare a T cell clone that proliferates indefinitely in culture.

In some aspects, the T cells are primary T cells. The term “primary Tcells” is intended to include T cells obtained from an individual, asopposed to T cells that have been maintained in culture for extendedperiods of time. Thus, primary T cells are particularly peripheral bloodT cells obtained from a subject. A population of primary T cells can becomposed of mostly one subset of T cells. Alternatively, the populationof primary T cells can be composed of different subsets of T cells.

The T cells can be from previously stored blood samples, from a healthyindividual, or alternatively from an individual affected with acondition. The condition can be an infectious disease, such as acondition resulting from a viral infection, a bacterial infection or aninfection by any other microorganism, or a hyperproliferative disease,such as cancer like melanoma. In a specific embodiment, the T cells arefrom an individual infected with a human immunodeficiency virus (HIV).In yet another embodiment, the T cells are from a subject suffering fromor susceptible to an autoimmune disease or T-cell pathologies. The Tcells can be of human origin, murine origin or any other mammalianspecies.

Methods of obtaining populations of cells comprising T cells are wellknown in the art. For example, peripheral blood mononuclear cells (PBMC)can be obtained as described according to methods known in the art.Examples of such methods are set forth in the Examples and is discussedby Kim et al. (1992); Biswas et al. (1990); Biswas et al. (1991).

In some aspects, the starting population of blood cells compriseshematopoietic stem cells (HSCs). HSCs normally reside in the bone marrowbut can be forced into the blood, a process termed mobilization usedclinically to harvest large numbers of HSCs in peripheral blood. Onemobilizing agent of choice is granulocyte colony-stimulating factor(G-CSF). CD34⁺ hematopoietic stem cells or progenitors that circulate inthe peripheral blood can be collected by apheresis techniques either inthe unperturbed state, or after mobilization following the externaladministration of hematopoietic growth factors like G-CSF. The number ofthe stem or progenitor cells collected following mobilization is greaterthan that obtained after apheresis in the unperturbed state. In someaspects, the source of the cell population is a subject whose cells havenot been mobilized by extrinsically applied factors because there is noneed to enrich hematopoietic stem cells or progenitor cells.

Methods of obtaining hematopoietic precursor cells from populations ofcells are also well known in the art. Hematopoietic precursor cells maybe expanded using various cytokines, such as hSCF, hFLT3, and/or IL-3(Akkina et al., 1996), or CD34⁺ cells may be enriched using MACS orFACS. As mentioned above, negative selection techniques may also be usedto enrich CD34⁺ cells.

Populations of cells for use in the methods described herein may bemammalian cells, such as human cells, non-human primate cells, rodentcells (e.g., mouse or rat), bovine cells, ovine cells, porcine cells,equine cells, sheep cells, canine cells, and feline cells or a mixturethereof. Non-human primate cells include rhesus macaque cells. The cellsmay be obtained from an animal, e.g., a human patient, or they may befrom cell lines. If the cells are obtained from an animal, they may beused as such, e.g., as unseparated cells (i.e., a mixed population);they may have been established in culture first, e.g., bytransformation; or they may have been subjected to preliminarypurification methods. For example, a cell population may be manipulatedby positive or negative selection based on expression of cell surfacemarkers; stimulated with one or more antigens in vitro or in vivo;treated with one or more biological modifiers in vitro or in vivo; or acombination of any or all of these. In an illustrative embodiment, acell population is subjected to negative selection for depletion ofnon-T cells and/or particular T cell subsets. Negative selection can beperformed on the basis of cell surface expression of a variety ofmolecules, including B cell markers such as CD19, and CD20; monocytemarker CD14; the NK cell marker CD56. Alternately, a cell population maybe subjected to negative selection for depletion of non-CD34⁺hematopoietic cells and/or particular hematopoietic cell subsets.Negative selection can be performed on the basis of cell surfaceexpression of a variety of molecules, such as a cocktail of antibodies(e.g., CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, CD235a, andCD41 (e.g., for cells of megakaryocyte lineage) which may be used forseparation of other cell types, e.g., via MACS or column separation.

It is also possible to obtain a cell sample from a subject, and then toenrich it for a desired cell type. For example, PBMCs and/or CD34⁺hematopoietic cells can be isolated from blood as described herein.Counter-flow centrifugation (elutriation) can be used to enrich for Tcells from PBMCs. Cells can also be isolated from other cells using avariety of techniques, such as isolation and/or activation with anantibody binding to an epitope on the cell surface of the desired celltype, for example, some T-cell isolation kits use antibody conjugatedbeads to both activate the cells and then allow column separation withthe same beads. Another method that can be used includes negativeselection using antibodies to cell surface markers to selectively enrichfor a specific cell type without activating the cell by receptorengagement.

Bone marrow cells may be obtained from iliac crest, femora, tibiae,spine, rib or other medullary spaces. Bone marrow may be taken out ofthe patient and isolated through various separations and washingprocedures. A known procedure for isolation of bone marrow cellscomprises the following steps: a) centrifugal separation of bone marrowsuspension in three fractions and collecting the intermediate fraction,or buffycoat; b) the buffycoat fraction from step (a) is centrifuged onemore time in a separation fluid, commonly Ficoll (a trademark ofPharmacia Fine Chemicals AB), and an intermediate fraction whichcontains the bone marrow cells is collected; and c) washing of thecollected fraction from step (b) for recovery of re-transfusable bonemarrow cells.

If one desires to use a population of cells enriched in T cells, suchpopulations of cells can be obtained from a mixed population of cells byleukapheresis and mechanical apheresis using a continuous flow cellseparator. For example, T cells can be isolated from the buffy coat byany known method, including separation over Ficoll-Hypaque™ gradient,separation over a Percoll gradient, or elutriation.

In certain aspects, T cells are activated by agents that bind to T cellreceptors to trigger a signaling cascade for T cell activation. Forexample, a CD3 antibody may be used. For T cell expansion to asignificant number and a proliferating state for reprogramming, acytokine may also be used, such as IL-2. In a certain aspect, bothanti-CD3 and anti-CD28 may be used for T cell activation whereco-stimulation is involved. In an alternative aspect, cross-linking ofthe anti-CD3 may be applied, such as plate bound anti-CD3. If solubleanti-CD3 is used to activate T cells in PBMC, the soluble anti-CD3antibody may bind to APCs in the PBMC, which then presents the antibodyto the T cells. If the soluble anti-CD3 antibody alone is used in apopulation of purified T-cells, anergy would result for the reasonsmentioned above. A certain embodiment comprises culturing T cells in thepresence of the anti-CD3 (OKT3) and IL2, which is advantageous andconvenient because there is no need to use costly and cumbersome beadsor plate-bound antibody; after adding OKT3 and IL2, the cellular milieuof PBMCs would help activate the T cells. The T cells then overcrowd theother cell types in the PBMC culture due to preferential expansion.

In certain aspects, the starting population of blood cells compriseslymphoblastoid cells, such as from lymphoblastoid cells lines (LCLs).Generation of LCLs is known in the art, for example, by infection of Bcells with Epstein-Barr virus (EBV) (Frisan et al., Epstein-Barr VirusProtocols, Part III, 125-127, 2001).

B. Reprogramming Factors

In certain embodiments, the starting population of somatic cells isreprogrammed to iPS cells by the introduction of reprogramming factors.The generation of iPS cells is crucial on the reprogramming factors usedfor the induction. The following factors or combination thereof could beused in the methods disclosed in the present disclosure. In certainaspects, nucleic acids encoding Sox and Oct (particularly Oct3/4) willbe included into the reprogramming vector. For example, one or morereprogramming vectors may comprise expression cassettes encoding Sox2,Oct4, Nanog and optionally Lin28, or expression cassettes encoding Sox2,Oct4, Klf4 and optionally c-Myc, or expression cassettes encoding Sox2,Oct4, and optionally Esrrb, or expression cassettes encoding Sox2, Oct4,Nanog, Lin28, Klf4, c-Myc, and optionally SV40 Large T antigen. Nucleicacids encoding these reprogramming factors may be comprised in the sameexpression cassette, different expression cassettes, the samereprogramming vector, or different reprogramming vectors.

Oct4 and certain members of the Sox gene family (Sox1, Sox2, Sox3, andSox15) have been identified as crucial transcriptional regulatorsinvolved in the induction process whose absence makes inductionimpossible. Additional genes, however, including certain members of theKlf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (c-Myc, L-Myc,and N-Myc), Nanog, and Lin28, have been identified to increase theinduction efficiency.

Oct4 (Pou5f1) is one of the family of octamer (“Oct”) transcriptionfactors, and plays a crucial role in maintaining pluripotency. Theabsence of Oct4 in Oct4⁺ cells, such as blastomeres and embryonic stemcells, leads to spontaneous trophoblast differentiation, and presence ofOct4 thus gives rise to the pluripotency and differentiation potentialof embryonic stem cells. Various other genes in the “Oct” family,including Oct4's close relatives, Oct1 and Oct6, fail to elicitinduction, thus demonstrating the exclusiveness of Oct-4 to theinduction process.

The Sox family of genes is associated with maintaining pluripotencysimilar to Oct4, although it is associated with multipotent andunipotent stem cells in contrast with Oct4, which is exclusivelyexpressed in pluripotent stem cells. While Sox2 was the initial geneused for reprogramming induction, other genes in the Sox family havebeen found to work as well in the induction process. Sox1 yields iPScells with a similar efficiency as Sox2, and genes Sox3, Sox15, andSox18 also generate iPS cells, although with decreased efficiency.

In embryonic stem cells, Nanog, along with Oct4 and Sox2, is necessaryin promoting pluripotency. Therefore, it was surprising when Yamanaka etal. reported that Nanog was unnecessary for induction although Thomsonet al. has reported it is possible to generate iPS cells with Nanog asone of the factors.

Lin28 is an mRNA binding protein expressed in embryonic stem cells andembryonic carcinoma cells associated with differentiation andproliferation. Thomson et al. demonstrated it is a factor in iPSgeneration, although it is unnecessary.

Klf4 of the Klf family of genes was initially identified by Yamanaka etal., 2007 and confirmed by Jaenisch et al., 1988 as a factor for thegeneration of mouse iPS cells and was demonstrated by Yamanaka et al.,2007 as a factor for generation of human iPS cells. However, Thompson etal. reported that Klf4 was unnecessary for generation of human iPS cellsand in fact failed to generate human iPS cells. Klf2 and Klf4 were foundto be factors capable of generating iPS cells, and related genes Klf1and Klf5 did as well, although with reduced efficiency.

The Myc family of genes are proto-oncogenes implicated in cancer.Yamanaka et al., 2007 and Jaenisch et al., 1988 demonstrated that c-Mycis a factor implicated in the generation of mouse iPS cells and Yamanakaet al., 2007 demonstrated it was a factor implicated in the generationof human iPS cells. However, Thomson et al. and Yamanaka et al. reportedthat c-Myc was unnecessary for generation of human iPS cells. SV40 largeantigen may be used to reduce or prevent the cytotoxcity which may occurwhen c-Myc is expressed.

The reprogramming proteins used in the present disclosure can besubstituted by protein homologs with about the same reprogrammingfunctions. Nucleic acids encoding those homologs could also be used forreprogramming. Conservative amino acid substitutions are preferred—thatis, for example, aspartic-glutamic as polar acidic amino acids;lysine/arginine/histidine as polar basic amino acids;leucine/isoleucine/methionine/valine/alanine/glycine/proline asnon-polar or hydrophobic amino acids; serine/threonine as polar oruncharged hydrophilic amino acids. Conservative amino acid substitutionalso includes groupings based on side chains. For example, a group ofamino acids having aliphatic side chains is glycine, alanine, valine,leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine. Forexample, it is reasonable to expect that replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the properties of theresulting polypeptide. Whether an amino acid change results in afunctional polypeptide can readily be determined by assaying thespecific activity of the polypeptide.

C. Reprogramming of Somatic Cells

In certain aspects of the present disclosure, reprogramming factors areexpressed from expression cassettes comprised in one or more vectors,such as an integrating vector or an episomal vector. In a furtheraspect, reprogramming proteins could be introduced directly into somaticcells by protein transduction.

One of skill in the art would be well-equipped to construct a vectorthrough standard recombinant techniques (see, for example, Sambrook etal., 2001 and Ausubel et al., 1996, both incorporated herein byreference). Vectors include but are not limited to, plasmids, cosmids,viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs), such as retroviral vectors (e.g.derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV,MPSV, SNV etc), lentiviral vectors (e.g. derived from HIV-1, HIV-2, SIV,BIV, FIV etc.), adenoviral (Ad) vectors including replication competent,replication deficient and gutless forms thereof, adeno-associated viral(AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virusvectors, Epstein-Barr virus vectors, herpes virus vectors, vacciniavirus vectors, Harvey murine sarcoma virus vectors, murine mammary tumorvirus vectors, Rous sarcoma virus vectors.

1. Viral Vectors

Viral vectors may be provided in certain aspects of the presentdisclosure. In generating recombinant viral vectors, non-essential genesare typically replaced with a gene or coding sequence for a heterologous(or non-native) protein. A viral vector is a kind of expressionconstruct that utilizes viral sequences to introduce nucleic acid andpossibly proteins into a cell. The ability of certain viruses to infectcells or enter cells via receptor-mediated endocytosis, and to integrateinto host cell genomes and express viral genes stably and efficientlyhave made them attractive candidates for the transfer of foreign nucleicacids into cells (e.g., mammalian cells). Non-limiting examples of virusvectors that may be used to deliver a nucleic acid of certain aspects ofthe present disclosure are described below.

Retroviruses have promise as gene delivery vectors due to their abilityto integrate their genes into the host genome, transfer a large amountof foreign genetic material, infect a broad spectrum of species and celltypes, and be packaged in special cell-lines (Miller, 1992).

In order to construct a retroviral vector, a nucleic acid is insertedinto the viral genome in place of certain viral sequences to produce avirus that is replication-defective. In order to produce virions, apackaging cell line containing the gag, pol, and env genes—but withoutthe LTR and packaging components—is constructed (Mann et al., 1983).When a recombinant plasmid containing a cDNA, together with theretroviral LTR and packaging sequences, is introduced into a specialcell line (e.g., by calcium phosphate precipitation), the packagingsequence allows the RNA transcript of the recombinant plasmid to bepackaged into viral particles, which are then secreted into the culturemedium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983).The medium containing the recombinant retroviruses is then collected,optionally concentrated, and used for gene transfer. Retroviral vectorsare able to infect a broad variety of cell types. However, integrationand stable expression require the division of host cells (Paskind etal., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomeret al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136).

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell—wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat—is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference.

2. Episomal Vectors

The use of plasmid- or liposome-based extra-chromosomal (i.e., episomal)vectors may be also provided in certain aspects of the presentdisclosure. Such episomal vectors may include, e.g., oriP-based vectors,and/or vectors encoding a derivative of EBNA-1. These vectors may permitlarge fragments of DNA to be introduced unto a cell and maintainedextra-chromosomally, replicated once per cell cycle, partitioned todaughter cells efficiently, and elicit substantially no immune response.

In particular, EBNA-1, the only viral protein required for thereplication of the oriP-based expression vector, does not elicit acellular immune response because it has developed an efficient mechanismto bypass the processing required for presentation of its antigens onMHC class I molecules (Levitskaya et al., 1997). Further, EBNA-1 can actin trans to enhance expression of the cloned gene, inducing expressionof a cloned gene up to 100-fold in some cell lines (Langle-Rouault etal., 1998; Evans et al., 1997). Finally, the manufacture of suchoriP-based expression vectors is inexpensive.

Other extra-chromosomal vectors include other lymphotrophic herpesvirus-based vectors. Lymphotrophic herpes virus is a herpes virus thatreplicates in a lymphoblast (e.g., a human B lymphoblast) and becomes aplasmid for a part of its natural life-cycle. Herpes simplex virus (HSV)is not a “lymphotrophic” herpes virus. Exemplary lymphotrophic herpesviruses include, but are not limited to EBV, Kaposi's sarcoma herpesvirus (KSHV); Herpes virus saimiri (HS) and Marek's disease virus (MDV).Other sources of episome-base vectors are also contemplated, such asyeast ARS, adenovirus, SV40, or BPV.

One of skill in the art would be well-equipped to construct a vectorthrough standard recombinant techniques (see, for example, Maniatis etal., 1988 and Ausubel et al., 1994, both incorporated herein byreference).

Vectors can also comprise other components or functionalities thatfurther modulate gene delivery and/or gene expression, or that otherwiseprovide beneficial properties to the targeted cells. Such othercomponents include, for example, components that influence binding ortargeting to cells (including components that mediate cell-type ortissue-specific binding); components that influence uptake of the vectornucleic acid by the cell; components that influence localization of thepolynucleotide within the cell after uptake (such as agents mediatingnuclear localization); and components that influence expression of thepolynucleotide.

Such components also may include markers, such as detectable and/orselection markers that can be used to detect or select for cells thathave taken up and are expressing the nucleic acid delivered by thevector. Such components can be provided as a natural feature of thevector (such as the use of certain viral vectors that have components orfunctionalities mediating binding and uptake), or vectors can bemodified to provide such functionalities. A large variety of suchvectors are known in the art and are generally available. When a vectoris maintained in a host cell, the vector can either be stably replicatedby the cells during mitosis as an autonomous structure, incorporatedwithin the genome of the host cell, or maintained in the host cell'snucleus or cytoplasm.

3. Transposon-based System

In certain aspects, the delivery of programming factors can use atransposon-transposase system. For example, the transposon-transposasesystem could be the well known Sleeping Beauty, the Frog Princetransposon-transposase system (for a description of the latter, see,e.g., EP1507865), or the TTAA-specific transposon PiggyBac system.

Transposons are sequences of DNA that can move around to differentpositions within the genome of a single cell, a process calledtransposition. In the process, they can cause mutations and change theamount of DNA in the genome. Transposons were also once called jumpinggenes, and are examples of mobile genetic elements.

There are a variety of mobile genetic elements, and they can be groupedbased on their mechanism of transposition. Class I mobile geneticelements, or retrotransposons, copy themselves by first beingtranscribed to RNA, then reverse transcribed back to DNA by reversetranscriptase, and then being inserted at another position in thegenome. Class II mobile genetic elements move directly from one positionto another using a transposase to “cut and paste” them within thegenome.

In particular embodiments, the constructs (e.g., the multi-lineageconstruct) provided in the present disclosure use a PiggyBac expressionsystem. PiggyBac (PB) DNA transposons mobilize via a “cut-and-paste”mechanism whereby a transposase enzyme (PB transposase), encoded by thetransposon itself, excises and re-integrates the transposon at othersites within the genome. PB transposase specifically recognizes PBinverted terminal repeats (ITRs) that flank the transposon; it binds tothese sequences and catalyzes excision of the transposon. PB thenintegrates at TTAA sites throughout the genome, in a relatively randomfashion. For the creation of gene trap mutations (or adapted forgenerating transgenic animals), the transposase is supplied in trans onone plasmid and is co-transfected with a plasmid containing donortransposon, a recombinant transposon comprising a gene trap flanked bythe binding sites for the transposase (ITRs). The transposase willcatalyze the excision of the transposon from the plasmid and subsequentintegration into the genome. Integration within a coding region willcapture the elements necessary for gene trap expression. PB possessesseveral ideal properties: (1) it preferentially inserts within genes (50to 67% of insertions hit genes) (2) it exhibits no local hopping(widespread genomic coverage) (3) it is not sensitive to over-productioninhibition in which elevated levels of the transposase cause decreasedtransposition 4) it excises cleanly from a donor site, leaving no“footprint,” unlike Sleeping Beauty.

4. Regulatory Elements

Expression cassettes included in reprogramming vectors useful in thepresent disclosure preferably contain (in a 5′-to-3′ direction) aeukaryotic transcriptional promoter operably linked to a protein-codingsequence, splice signals including intervening sequences, and atranscriptional termination/polyadenylation sequence.

a. Promoter/Enhancers

The expression constructs provided herein comprise promoter to driveexpression of the programming genes. A promoter generally comprises asequence that functions to position the start site for RNA synthesis.The best known example of this is the TATA box, but in some promoterslacking a TATA box, such as, for example, the promoter for the mammalianterminal deoxynucleotidyl transferase gene and the promoter for the SV40late genes, a discrete element overlying the start site itself helps tofix the place of initiation. Additional promoter elements regulate thefrequency of transcriptional initiation. Typically, these are located inthe region 30-110 bp upstream of the start site, although a number ofpromoters have been shown to contain functional elements downstream ofthe start site as well. To bring a coding sequence “under the controlof” a promoter, one positions the 5′ end of the transcription initiationsite of the transcriptional reading frame “downstream” of (i.e., 3′ of)the chosen promoter. The “upstream” promoter stimulates transcription ofthe DNA and promotes expression of the encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include theβ-lactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated that the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al. 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination (as per, for example, theEukaryotic Promoter Data Base EPDB) could also be used to driveexpression. Use of a T3, T7 or SP6 cytoplasmic expression system isanother possible embodiment. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

Non-limiting examples of promoters include early or late viralpromoters, such as, SV40 early or late promoters, cytomegalovirus (CMV)immediate early promoters, Rous Sarcoma Virus (RSV) early promoters;eukaryotic cell promoters, such as, e. g., beta actin promoter (Ng,1989; Quitsche et al., 1989), GADPH promoter (Alexander et al., 1988,Ercolani et al., 1988), metallothionein promoter (Karin et al., 1989;Richards et al., 1984); and concatenated response element promoters,such as cyclic AMP response element promoters (cre), serum responseelement promoter (sre), phorbol ester promoter (TPA) and responseelement promoters (tre) near a minimal TATA box. It is also possible touse human growth hormone promoter sequences (e.g., the human growthhormone minimal promoter described at Genbank, accession no. X05244,nucleotide 283-341) or a mouse mammary tumor promoter (available fromthe ATCC, Cat. No. ATCC 45007).

Tissue-specific transgene expression, especially for reporter geneexpression in hematopoietic cells and precursors of hematopoietic cellsderived from programming, may be desirable as a way to identify derivedhematopoietic cells and precursors. To increase both specificity andactivity, the use of cis-acting regulatory elements has beencontemplated. For example, a hematopoietic cell-specific promoter may beused. Many such hematopoietic cell-specific promoters are known in theart, such as promoters of the hematopoietic genes provided in Table 1.

In certain aspects, methods of the present disclosure also concernenhancer sequences, i.e., nucleic acid sequences that increase apromoter's activity and that have the potential to act in cis, andregardless of their orientation, even over relatively long distances (upto several kilobases away from the target promoter). However, enhancerfunction is not necessarily restricted to such long distances as theymay also function in close proximity to a given promoter.

Many hematopoietic cell promoter and enhancer sequences have beenidentified, and may be useful in present methods. See, e.g., U.S. Pat.No. 5,556,954; U.S. Patent App. 20020055144; U.S. Patent App.20090148425.

b. Initiation Signals and Linked Expression

A specific initiation signal also may be used in the expressionconstructs provided in the present disclosure for efficient translationof coding sequences. These signals include the ATG initiation codon oradjacent sequences. Exogenous translational control signals, includingthe ATG initiation codon, may need to be provided. One of ordinary skillin the art would readily be capable of determining this and providingthe necessary signals. It is well known that the initiation codon mustbe “in-frame” with the reading frame of the desired coding sequence toensure translation of the entire insert. The exogenous translationalcontrol signals and initiation codons can be either natural orsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements.

In certain embodiments, internal ribosome entry sites (IRES) elementsare used to create multigene, or polycistronic, messages. IRES elementsare able to bypass the ribosome scanning model of 5′ methylated Capdependent translation and begin translation at internal sites (Pelletierand Sonenberg, 1988). IRES elements from two members of the picornavirusfamily (polio and encephalomyocarditis) have been described (Pelletierand Sonenberg, 1988), as well an IRES from a mammalian message (Macejakand Sarnow, 1991). IRES elements can be linked to heterologous openreading frames. Multiple open reading frames can be transcribedtogether, each separated by an IRES, creating polycistronic messages. Byvirtue of the IRES element, each open reading frame is accessible toribosomes for efficient translation. Multiple genes can be efficientlyexpressed using a single promoter/enhancer to transcribe a singlemessage (see U.S. Pat. Nos. 5,925,565 and 5,935,819, each hereinincorporated by reference).

Additionally, certain 2A sequence elements could be used to createlinked- or co-expression of programming genes in the constructs providedin the present disclosure. For example, cleavage sequences could be usedto co-express genes by linking open reading frames to form a singlecistron. An exemplary cleavage sequence is the F2A (Foot-and-mouthdisease virus 2A) or a “2A-like” sequence (e.g., Thosea asigna virus 2A;T2A) (Minskaia and Ryan, 2013). In particular embodiments, anF2A-cleavage peptide is used to link expression of the genes in themulti-lineage construct.

c. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), for example, anucleic acid sequence corresponding to oriP of EBV as described above ora genetically engineered oriP with a similar or elevated function inprogramming, which is a specific nucleic acid sequence at whichreplication is initiated. Alternatively a replication origin of otherextra-chromosomally replicating virus as described above or anautonomously replicating sequence (ARS) can be employed.

d. Selection and Screenable Markers

In certain embodiments, cells containing a nucleic acid construct may beidentified in vitro or in vivo by including a marker in the expressionvector. Such markers would confer an identifiable change to the cellpermitting easy identification of cells containing the expressionvector. Generally, a selection marker is one that confers a propertythat allows for selection. A positive selection marker is one in whichthe presence of the marker allows for its selection, while a negativeselection marker is one in which its presence prevents its selection. Anexample of a positive selection marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selection markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes as negative selection markers such as herpes simplex virusthymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may beutilized. One of skill in the art would also know how to employimmunologic markers, possibly in conjunction with FACS analysis. Themarker used is not believed to be important, so long as it is capable ofbeing expressed simultaneously with the nucleic acid encoding a geneproduct. Further examples of selection and screenable markers are wellknown to one of skill in the art.

Introduction of a nucleic acid, such as DNA or RNA, into the pluripotentstem cells to be programmed to hematopoietic precursor cells with thecurrent disclosure may use any suitable methods for nucleic aciddelivery for transformation of a cell, as described herein or as wouldbe known to one of ordinary skill in the art. Such methods include, butare not limited to, direct delivery of DNA such as by ex vivotransfection (Wilson et al., 1989, Nabel et al, 1989), by injection(U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524,5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated hereinby reference), including microinjection (Harland and Weintraub, 1985;U.S. Pat. No. 5,789,215, incorporated herein by reference); byelectroporation (U.S. Pat. No. 5,384,253, incorporated herein byreference; Tur-Kaspa et al., 1986; Potter et al., 1984); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE-dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al., 1987); by liposome mediated transfection (Nicolau and Sene,1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991) and receptor-mediatedtransfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectilebombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat.Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880,and each incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

III. Production of Immune Cells

A. Production of HPCs

Embodiments of the present disclosure concern the differentiation ofsomatic cell-derived PSCs to HPCs. The somatic cell-derived PSCs can bedifferentiated into HPCs by methods known in the art such as describedin U.S. Pat. No. 8,372,642, which is incorporated by reference herein.For example, combinations of BMP4, VEGF, Flt3 ligand, IL-3, and GM-CSFmay be used to promote hematopoietic differentiation. In certainembodiments, the sequential exposure of cell cultures to a first mediato prepare PSCs for differentiation, a second media that includes BMP4,VEGF, and FGF, followed by culture in a third media that includes Flt3ligand, SCF, TPO, IL-3, and IL-6 can differentiate pluripotent cellsinto hematopoietic precursor cells and hematopoietic cells. The seconddefined media can also comprise heparin. Further, inclusion of FGF-2 (50ng/ml) in the media containing BMP4 and VEGF can enhance the efficiencyof the generation of hematopoietic precursor cells from pluripotentcells. In addition, inclusion of a Glycogen synthase kinase 3 (GSK3)inhibitor (e.g., CHIR99021, BIO, and SB-216763) in the first definedmedia can further enhance the production of HPCs.

Differentiation of pluripotent cells into hematopoietic precursor cellsmay be performed using defined or undefined conditions. Generally, itwill be appreciated that defined conditions are generally preferable inembodiments where the resulting cells are intended to be administered toa human subject. Hematopoietic stem cells may be derived frompluripotent stem cells under defined conditions (e.g., using a TeSRmedia), and hematopoietic cells may be generated from embryoid bodiesderived from pluripotent cells. In other embodiments, pluripotent cellsmay be co-cultured on OP9 cells or mouse embryonic fibroblast cells andsubsequently differentiated.

Pluripotent cells may be allowed to form embryoid bodies or aggregatesas a part of the differentiation process. The formation of “embryoidbodies” (EBs), or clusters of growing cells, in order to inducedifferentiation generally involves in vitro aggregation of humanpluripotent stem cells into EBs and allows for the spontaneous andrandom differentiation of human pluripotent stem cells into multipletissue types that represent endoderm, ectoderm, and mesoderm origins.Three-dimensional EBs can thus be used to produce some fraction ofhematopoietic cells and endothelial cells.

EBs may be formed using the following protocol. Undifferentiated iPSCsadapted to feeder free growth on Matrigel™ coated plates may beharvested at confluency using 0.5M EDTA treatment for about 8-10 minutesat room temperature. The EDTA is aspirated after the incubation and theEBs may be formed by collecting the cells in SFD media containing rockinhibitor or blebbistatin. The media may be changed the next day to EB1differentiation media containing different cytokine formulations. Thecells are plated at a density of 0.25-0.5 million cells per ml topromote aggregate formation.

To promote aggregate formation, the cells may be transferred tolow-attachment plates for an overnight incubation in serum-freedifferentiation (SFD) medium, consisting of 75% IMDM (Gibco), 25% Ham'sModified F12 (Cellgro) supplemented with 0.05% N2 and B-27 without RAsupplements, 200 mM 1-glutamine, 0.05 mg/ml Ascorbic Acid-2-phosphateMagnesium Salt (Asc 2-P) (WAKO), and 4.5×10⁻⁴ MTG. The next day thecells may be collected from each well and centrifuged. The cells maythen be resuspended in “EB differentiation media,” which consists of SFDbasal media supplemented with about 50 ng/ml bone morphogenetic factor(BMP-4), about 50 ng/ml vascular endothelial growth factor (VEGF), and50 ng/ml zb FGF for the first four days of differentiation. The cellsare half fed ever 48 hrs. On the fifth day of differentiation the mediais replaced with a second media comprised of SFD media supplemented with50 ng/ml stem cell factor (SCF), about 50 ng/ml Flt-3 ligand (Flt-3L),50 ng/ml interleukin-6 (IL-6), 50 ng/ml interleukin-3 (IL-3), 50 ng/mlthrombopoieitin (TPO). The cells are half fed every 48 hrs with freshdifferentiation media. The media changes are performed by spinning downthe differentiation cultures at 300 g for 5 minutes and aspirating halfthe volume from the differentiating cultures and replenishing it withfresh media. In certain embodiments, the EB differentiation media mayinclude about BMP4 (e.g., about 50 ng/ml), VEGF (e.g., about 50 ng/ml),and optionally FGF-2 (e.g., about 25-75 ng/ml or about 50 ng/ml). Thesupernatant may be aspirated and replaced with fresh differentiationmedium. Alternately the cells may be half fed every two days with freshmedia. The cells may be harvested at different time points during thedifferentiation process.

Hematopoietic precursor cells may be cultured from pluripotent stemcells using a defined medium. Methods for the differentiation ofpluripotent cells into hematopoietic CD34⁺ stem cells using a definedmedia are described, e.g., in U.S. application Ser. No. 12/715,136 whichis incorporated by reference in its entirety without disclaimer. It isanticipated that these methods may be used with the present disclosure.

For example, a defined medium may be used to induce hematopoietic CD34⁺differentiation. The defined medium may contain the growth factorsBMP-4, VEGF, Flt3 ligand, IL-3 and/or GMCSF. Pluripotent cells may becultured in a first defined media comprising BMP4, VEGF, and optionallyFGF-2, followed by culture in a second media comprising either (Flt3ligand, IL-3, and GMCSF) or (Flt3 ligand, IL-3, IL-6, and TPO). Thefirst and second media may also comprise one or more of SCF, IL-6,G-CSF, EPO, FGF-2, and/or TPO. Substantially hypoxic conditions (e.g.,less than 20% 02) may further promote hematopoietic or endothelialdifferentiation.

Cells may be substantially individualized via mechanical or enzymaticmeans (e.g., using a trypsin or TrypLE™). A ROCK inhibitor (e.g., H1152or Y-27632) may also be included in the media. It is anticipated thatthese approaches may be automated using, e.g., robotic automation.

In certain embodiments, substantially hypoxic conditions may be used topromote differentiation of pluripotent cells into hematopoieticprogenitor cells. As would be recognized by one of skill in the art, anatmospheric oxygen content of less than about 20.8% would be consideredhypoxic. Human cells in culture can grow in atmospheric conditionshaving reduced oxygen content as compared to ambient air. This relativehypoxia may be achieved by decreasing the atmospheric oxygen exposed tothe culture media. Embryonic cells typically develop in vivo underreduced oxygen conditions, generally between about 1% and about 6%atmospheric oxygen, with carbon dioxide at ambient levels. Withoutwishing to be bound by theory, it is anticipated that hypoxic conditionsmay mimic an aspect of certain embryonic developmental conditions. Asshown in the below examples, hypoxic conditions can be used in certainembodiments to promote additional differentiation of pluripotent cells,such as iPSC or hESC, into a more differentiated cell type, such ashematopoietic precursor cells.

The following hypoxic conditions may be used to promote differentiationof pluripotent cells into hematopoietic progenitor cells. In certainembodiments, an atmospheric oxygen content of less than about 20%, lessthan about 19%, less than about 18%, less than about 17%, less thanabout 16%, less than about 15%, less than about 14%, less than about13%, less than about 12%, less than about 11%, less than about 10%, lessthan about 9%, less than about 8%, less than about 7%, less than about6%, less than about 5%, about 5%, about 4%, about 3%, about 2%, or about1% may be used to promote differentiation into hematopoietic precursorcells. In certain embodiments, the hypoxic atmosphere comprises about 5%oxygen gas.

Regardless of the specific medium being used in any given hematopoieticprogenitor cell expansion, the medium used is preferably supplementedwith at least one cytokine at a concentration from about 0.1 ng/mL toabout 500 ng mL, more usually 10 ng/mL to 100 ng/mL. Suitable cytokines,include but are not limited to, c-kit ligand (KL) (also called steelfactor (StI), mast cell growth factor (MGF), and stem cell factor(SCF)), IL-6, G-CSF, IL-3, GM-CSF, IL-1α, IL-11 MIP-1α, LIF, c-mplligand/TPO, and flk2/flk3 ligand (Flt2L or Flt3L). (Nicola et al., 1979;Golde et al., 1980; Lusis, 1981; Abboud et al., 1981; Okabe, 1982;Fauser et al., 1981). Particularly, the culture will include at leastone of SCF, Flt3L and TPO. More particularly, the culture will includeSCF, Flt3L and TPO.

In one embodiment, the cytokines are contained in the media andreplenished by media perfusion. Alternatively, when using a bioreactorsystem, the cytokines may be added separately, without media perfusion,as a concentrated solution through separate inlet ports. When cytokinesare added without perfusion, they will typically be added as a 10× to100× solution in an amount equal to one-tenth to 1/100 of the volume inthe bioreactors with fresh cytokines being added approximately every 2to 4 days. Further, fresh concentrated cytokines also can be addedseparately in addition, to cytokines in the perfused media.

In some embodiments, the HPCs exhibit disrupted Methyl-CpG BindingProtein 2 (MeCP2) and are cultured under conditions to promote myeloiddifferentiation or lymphoid differentiation. In some aspects, the HPCsexpress a non-functional MeCP2 that has essentially no binding tomethylated DNA. In certain aspects, the HPCs do not express MeCP2 atlevels that are sufficient to effect MeCP2 DNA binding activity. Inparticular aspects, the MeCP2 is non-functional by virtue of atruncation or mutation in the MeCP2 gene. In some aspects, obtainingHPCs that exhibit disrupted MeCP2 comprises contacting the HPCs withsiRNA, shRNA or a small molecule inhibitor of MeCP2.

B. Lymphoid Cell Differentiation

The HPCs which are differentiated from the somatic cell-derived PSCs canthen be further differentiated to lymphoid lineage cells, including Tcells, NK cells, and T/NK cells. In some aspects, HPCs duringdifferentiation are isolated at Day 7-12, such as Day 8-11, fordifferentiation to lymphoid cells. The HPCs at this stage may beidentified by expression of CD34 and CD43. In addition the HPCs withlymphoid potential can express CD144, DLL4, CD7 and CD235 at low levelswhich decline at Day 11, implying that a certain threshold level ofexpression of these markers is needed to prime cells towards lymphoiddifferentiation in the presence of DLL4.

In some aspects, HPCs isolated at day 7-11, such as day 7, day 8, day 9,day 10 or day 11 of the differentiation process can be differentiated tolymphoid cells such as T and NK cells. In some aspects, the timing ofthe origin for lymphoid progenitors coincides with the decline ofhematoendothelial progenitors and the emergence of erythroid progenitorsduring HPC differentiation. In particular aspects, Day 9 HPCs may havean increased efficiency at generating T cells. HPCs capable of lymphoiddifferentiation can be isolated and/or identified by the expression ofcertain markers. For example, cells with surface expression of CD34and/or CD43 may be selected for lymphoid differentiation. Additionalmarkers for detecting lymphoid progenitors include DLL4, CD144, CD31,CD34, CD43^(lo), CD45^(lo/−), CD235, CD7, Flk-1, APNLR. In particularaspects, the presence of CD34/CD7, CD235/CD7, DLL4/CD34, DLL4/CD31,DLL4/CD144, or CD34/CD43^(lo) double positive populations is used toidentify lymphoid progenitors. CD144 expression on HPCs co stains withCD31, CD34 and DLL4. CD7 expression on HPCs co-stains with CD235, CD34and CD43. Hence HPCs co-expressing CD144 and CD7 demonstrate lymphoidpotential capture precursors expressing membrane bound notch ligand(DLL4) along with hematoendothelial markers and create the phenotypicsignature for emerging lymphoid progenitors capable of generatinglineages of definitive hematopoiesis in vitro. In particular aspects,the HPCs may be further sorted into cells with enhanced lymphoidpotential by sorting of the surface markers including CD31, CD34, CD144,CD43, CD45, CD6, CD335, Flk-1, and DLL4. In some aspects, the positivefractions of CD114/CD34, CD144/CD45, CD144/CD7, and CD144/CD34/CD45/CD7of HPCs are differentiated to lymphoid cells. In particular aspects, theCD144/CD7 positive fractions of HPCs is differentiated to lymphoidcells.

The HPCs may be cultured in defined, feeder free conditions for lymphoiddifferentiation. A culture media may contain one or more matrixcomponents, such as RetroNectin, fibronectin or a RGD peptide. Withoutwishing to be bound by any theory, a matrix component may provide asolid support for the growth of embryonic stem cells. In certainembodiments, a matrix component may be applied to a culturing surfaceand contacted with culture media prior to seeding cells into the media.For example, cells may be cultured in a defined media (e.g., a TeSRmedia) on plates coated with fibronectin or Matrigel™ prior tomechanically separating the cells into clumps or individualizing cellsand inducing differentiation into hematopoietic precursor cells.

Various matrix components may be used to culture pluripotent cellsincluding a collagen (e.g., collagen IV), laminin, vitronectin,Matrigel™, gelatin, polylysine, thrombospondin (e.g., TSP-1, -2, -3, -4and/or -5), and/or ProNectin-F™. In certain embodiments, the use of onlyMatrigel™, collagen IV, or laminin with cells previously cultured usingTeSR may be avoided due to possible adverse effects on cell viability;nonetheless, these compositions may be advantageously used incombination with other matrix components. Combinations of these matrixcomponents may provide additional benefit for promoting cell growth andcell viability. In certain embodiments, 1, 2, 3, 4, 5, 6, or more of theabove matrix components may be used to culture cells, e.g., prior tohematopoietic differentiation.

An exemplary feeder free matrix for lymphoid differentiation isdisclosed in Example 4. In particular aspects, a nontissueculture-treated plate may be coated with DLL4:Fc chimera protein andRetroNectin (fibronectin fragment CH-296; Takara Shuzo, Japan) for usein lymphoid differentiation of HPCs.

In some embodiments, ascorbic acid may be used to enhance lymphoiddifferentiation. The defined media may be supplemented with about 10 μMto about 1 mM ascorbic acid, such as about 50 μM to about 100 μM, suchas about 95 μM. The ascorbic acid may be selected from variousascorbates, such as ascorbic acid magnesium phosphate. In someembodiments, nicotinamide (e.g., nicotinic acid) may be used to enhancelymphoid differentiation, such as at a concentration of about 0.1 mM toabout 5 mM.

In some aspects, the HPCs are differentiated to lymphoid cells, such asT cells, by altering the endogenous activity of a Notch ligand byadministering a substance that increases the production of the Notchligand in a subject. The method also includes culturing the cells in amedium, wherein the medium includes an effective amount of a notchligand and one or more cytokines selected from the group consisting ofIL-7, IL-15, SCF, Flt-3 and IL-3. In some particular embodiments, themedium can further include IL-6. In some embodiments, the notch ligandis delta4 notch ligand (DLL4), such as DLL4:Fc chimera.

A Notch ligand is selected that promotes and maintains differentiationand proliferation of cells of the T cell lineage. A Notch ligand may behuman in origin, or may be derived from other species, includingmammalian species such as rodent, dog, cat, pig, sheep, cow, goat, andprimates. Particular examples of Notch Ligands include the Delta family.The Delta family includes Delta-1 (Genbank Accession No. AF003522, Homosapiens), Delta-3 (Genbank Accession No. AF084576, Rattus norvegicus),Delta-like 1 (Genbank Accession No. NM_005618 and NP_005609, Homosapiens; Genbank Accession No. X80903, 148324, M. musculus), Delta-like3 (Genbank Accession No. NM_053666, N_446118, Rattus norvegicus),Delta-4 (Genbank Accession No. AF273454, BAB18580, Mus musculus; GenbankAccession No. AF279305, AAF81912, Homo sapiens), and Delta-like 4(Genbank Accession. No. Q9NR61, AAF76427, AF253468, NM_019074, Homosapiens; Genbank Accession No. NM_019454, mus musculus). Notch ligandsare commercially available or can be produced by recombinant DNAtechniques and purified to various degrees.

The method further includes the step of maintaining the HPC cells in theculture described above for a duration of time sufficient to producedifferentiated NK cells. In some embodiments, differentiated NK cellsemerge in the cultures along with T cells, however the NK cells maycease to proliferate after week 6. In general, the determination of anincrease in the number of NK cells and/or their state of differentiationis assessed using conventional methods known to those of ordinary skillin the art. For example, the cultured cells may be monitored by flowcytometry for the development of NK cells by staining the cells withanti-CD56 and anti-CD3 antibodies. Cells which are CD56⁺/CD3⁻ would beindicative of differentiated NK cells.

C. Myeloid Differentiation

HPCs produced from somatic cell-derived PSCs may be differentiated intomyeloid cells using, e.g., a myeloid differentiation medium. A myeloiddifferentiation medium may be a serum-free or defined medium, and themedium may contain SCF, EPO, TPO, insulin, dexamethasone orhydrocortisone, and/or transferrin. The myeloid cells may be dendriticcells, macrophages, neutrophils, monocytes, basophils, neutrophils, mastcells, and/or eosinphils. In particular aspects, the myeloid cells aredendritic cells. Exemplary myeloid differentiation and expansion mediumare described, for example, in Tables 4-6.

In one exemplary method, HPCs are transferred in low attachment platesto a medium containing SFEM (Stem Cell Technologies), heparin (e.g., 1to 10 U/mL, such as 5 U/mL, Sigma), TPO (e.g., 50 to 150 ng/mL, such as100 ng/mL), human recombinant SCF (e.g., 50 to 150 ng/mL, such as 100ng/mL), FLT3L (e.g., 50 to 150 ng/mL, such as 100 ng/mL), IL-3 (e.g., 1to 20 ng/mL, such as 10 ng/mL), and IL-6 (e.g., 1 to 20 ng/mL, such as10 ng/mL). After about 5-15 days, such as 8 days, myeloid cells areexpanded in SFEM medium containing GM-CSF (e.g., 25 to 150 ng/mL, suchas 100 ng/mL). Finally, the cells are cultured in a medium containingSFEM (Stem Cell Technologies), Excyte (e.g., 0.1% to 2%, such as 1%),GM-CSF (25 to 150 ng/mL, such as 100 ng/mL), IL-4 (10 to 30 ng/mL, suchas 20 ng/mL), and TNFα (0.5 to 5 ng/mL, such as 2.5 ng/mL), for anadditional 1-2 weeks to produce dendritic cells. The dendritic cells canbe characterized by expression of one or more markers selected from thegroup consisting of CD209⁺, CD1a⁺, HLA-DR⁺, CD11c⁺, CD14⁺, CD83⁺, andCD86⁺. These markers predominantly stain myeloid DCs and notplasmocytoid DCs (CD123⁺). Wright staining can be performed on cytospinsamples to confirm the classic morphology of dendritic cells.

D. Cell Culture

In certain embodiments, substantially hypoxic conditions may be used topromote differentiation of HPCs to myeloid or lymphoid lineages. Incertain embodiments, an atmospheric oxygen content of less than about20%, less than about 19%, less than about 18%, less than about 17%, lessthan about 16%, less than about 15%, less than about 14%, less thanabout 13%, less than about 12%, less than about 11%, less than about10%, less than about 9%, less than about 8%, less than about 7%, lessthan about 6%, less than about 5%, about 5%, about 4%, about 3%, about2%, or about 1% may be used to promote differentiation intohematopoietic precursor cells. In certain embodiments, the hypoxicatmosphere comprises about 5% oxygen gas.

As described herein, one or more defined culture medium may beadvantageously used to promote the differentiation of HPCs to myeloidand lymphoid lineages; in particular, the elimination of animal productssuch as serum and mouse feeder layers can reduce the risks associatedwith exposure of cells to animal products and allow for the generationof cells that could be more safely administered to a human subject. Astraditional stem cell culture development has relied on serum productsand mouse feeder layers for differentiating stem cells into a variety ofcell types, these traditional procedures have limited the scale on whichdifferentiation can be conducted, increased biological variability andpotential contamination, and severely hampered the use of ES cells intranslational therapies in which they might otherwise prove useful.

Generally, cells of the present disclosure are cultured in a culturemedium, which is a nutrient-rich buffered solution capable of sustainingcell growth. Culture media suitable for isolating, expanding anddifferentiating pluripotent stem cells into hematopoietic precursorcells and hematopoietic cells according to the method described hereininclude but not limited to high glucose Dulbecco's Modified Eagle'sMedium (DMEM), DMEM/F-15, RPMI 1640, Iscove's modified Dubelcco's media(IMDM), and Opti-MEM SFM (Invitrogen Inc.). Chemically Defined Mediumcomprises a minimum essential medium such as Iscove's ModifiedDulbecco's Medium (IMDM) (Gibco), supplemented with human serum albumin,human ExCyte lipoprotein, transferrin, insulin, vitamins, essential andnon-essential amino acids, sodium pyruvate, glutamine and a mitogen isalso suitable. As used herein, a mitogen refers to an agent thatstimulates division of a cell. An agent can be a chemical, usually someform of a protein that encourages a cell to commence cell division,triggering mitosis. In one embodiment, serum free media such as thosedescribed in U.S. Ser. No. 08/464,599 and WO 96/39487, and the “completemedia” as described in U.S. Pat. No. 5,486,359 are contemplated for usewith methods described herein. In some embodiments, the culture mediumis supplemented with 10% Fetal Bovine Serum (FBS), human autologousserum, human AB serum or platelet rich plasma supplemented with heparin(2 U/ml).

Immune cells can be generated by culturing pluripotent stem cells orhematopoietic precursor cells in a medium under conditions that increasethe intracellular level of factors sufficient to promote differentiationof the cells into myeloid or lymphoid lineages. The medium may alsocontain one or more hematopoietic cell differentiation and maturationagents, like various kinds of growth factors. These agents may eitherhelp induce cells to commit to a more mature phenotype—or preferentiallypromote survival of the mature cells—or have a combination of both ofthese effects. Differentiation and maturation agents may include solublegrowth factors (peptide hormones, cytokines, ligand-receptor complexes,and other compounds) that are capable of promoting the growth of cellsof the hematopoietic cell lineage. Non-limiting examples of such agentsinclude but are not limited to hematopoietic or endothelial growthfactors such as fibroblast growth factor (FGF), vascular endothelialgrowth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO),FLT-3 ligand (FLT3L), interleukin-3 (IL-3), interleukin-6 (IL-6),interleukin-9 (IL-9), or granulocyte colony-stimulating factor (G-CSF),or isoforms or variants thereof.

IV. Uses of Immune Cells

The immune cells provided by methods and compositions of certain aspectscan be used in a variety of applications. These include but are notlimited to transplantation or implantation of the cells in vivo;screening cytotoxic compounds, carcinogens, mutagens growth/regulatoryfactors, pharmaceutical compounds, etc., in vitro; elucidating themechanism of hematological diseases and injuries; studying the mechanismby which drugs and/or growth factors operate; diagnosing and monitoringcancer in a patient; gene therapy; and the production of biologicallyactive products, to name but a few.

A. Test Compound Screening

Immune cells of this disclosure can be used to screen for factors (suchas solvents, small molecule drugs, peptides, and polynucleotides) orenvironmental conditions (such as culture conditions or manipulation)that affect the characteristics of lymphoid cells provided herein.

Particular screening applications of this disclosure relate to thetesting of pharmaceutical compounds in drug research. The reader isreferred generally to the standard textbook In vitro Methods inPharmaceutical Research, Academic Press, 1997, and U.S. Pat. No.5,030,015. In certain aspects, myeloid and lymphoid cells play the roleof test cells for standard drug screening and toxicity assays, as havebeen previously performed on hematopoietic cells and precursors inshort-term culture. Assessment of the activity of candidatepharmaceutical compounds generally involves combining the hematopoieticcells or precursors provided in certain aspects with the candidatecompound, determining any change in the morphology, marker phenotype, ormetabolic activity of the cells that is attributable to the compound(compared with untreated cells or cells treated with an inert compound),and then correlating the effect of the compound with the observedchange. The screening may be done either because the compound isdesigned to have a pharmacological effect on hematopoietic cells orprecursors, or because a compound designed to have effects elsewhere mayhave unintended effects on hematopoietic cells or precursors. Two ormore drugs can be tested in combination (by combining with the cellseither simultaneously or sequentially), to detect possible drug-druginteraction effects.

B. Hematopoietic Cell Therapy

This disclosure also provides for the use of immune cells providedherein to restore a degree of function to a subject needing suchtherapy, perhaps due to a hematological disease or disorder or aninjury. For example, immune cells derived by methods disclosed hereinmay be used to treat hematological diseases and disorders such ashemoglobinopathies, anemias, etc. Such cells may be useful for thetreatment of hematopoietic cell deficiencies caused by cell-suppressivetherapies, such as chemotherapy.

To determine the suitability of cells provided herein for therapeuticapplications, the cells can first be tested in a suitable animal model.At one level, cells are assessed for their ability to survive andmaintain their phenotype in vivo. Cells provided herein are administeredto immunodeficient animals (such as NOG mice, or animals renderedimmunodeficient chemically or by irradiation) at a site amenable forfurther observation, such as under the kidney capsule, into the spleen,into a liver lobule, or into the bone marrow. Tissues are harvestedafter a period of a few days to several weeks or more, and assessed asto whether starting cell types such as erythrocytes are still present.This can be performed by providing the administered cells with adetectable label (such as green fluorescent protein, or(β-galactosidase); or by measuring a constitutive marker specific forthe administered human cells. Where cells provided herein are beingtested in a rodent model, the presence and phenotype of the administeredcells can be assessed by immunohistochemistry or ELISA usinghuman-specific antibody, or by RT-PCR analysis using primers andhybridization conditions that cause amplification to be specific forhuman polynucleotide sequences. Suitable markers for assessing geneexpression at the mRNA or protein level are provided elsewhere in thisdisclosure.

Immune cells provided by methods of the present disclosure may be testedin various animal models for their ability to treat hematologicaldisorders and injuries. For example, a sickle cell anemia mouse model orthe T/B cell-deficient Rag-2 knockout mouse may be particularly usefulanimal models for testing the myeloid and lymphoid cells disclosedherein.

Immune cells provided in certain aspects of the present disclosure thatdemonstrate desirable functional characteristics or efficacy in animalmodels, may also be suitable for direct administration to human subjectsin need thereof. For purposes of hemostasis, the cells can beadministered at any site that has adequate access to the circulation.Hematopoietic cells or precursors thereof may also be delivered at asite of injury or disease.

The cells provided in certain aspects of this present disclosure can beused for therapy of any subject in need thereof. Human conditions thatmay be appropriate for such therapy include the various anemias andhemoglobinopathies, as well as diseases characterized by decreasednumbers of hematopoietic cells (such as, for example, myelodysplasticsyndrome, myelofibrosis, neutropenia, agranulocytosis, Glanzmann'sthrombasthenia, thrombocytopenia, and acquired immune deficiencysyndrome). For human therapy, the dose is generally between about 10⁹and 10¹² cells, and typically between about 5×10⁹ and 5×10¹⁰ cells,making adjustments for the body weight of the subject, nature andseverity of the affliction, and the replicative capacity of theadministered cells. The ultimate responsibility for determining the modeof treatment and the appropriate dose lies with the managing clinician.

C. Distribution for Commercial, Therapeutic, and Research Purposes

For purposes of manufacture, distribution, and use, the immune cells ofthis disclosure are typically supplied in the form of a cell culture orsuspension in an isotonic excipient or culture medium, optionally frozento facilitate transportation or storage.

Also provided herein are different reagent systems, comprising a set orcombination of cells that exist at any time during manufacture,distribution, or use. The cell sets comprise any combination of two ormore cell populations described in this disclosure, exemplified but notlimited to programming-derived cells (hematopoietic lineage cells, theirprecursors and subtypes), in combination with undifferentiated stemcells, somatic cell-derived hematopoietic cells, or other differentiatedcell types. The cell populations in the set sometimes share the samegenome or a genetically modified form thereof. Each cell type in the setmay be packaged together, or in separate containers in the samefacility, or at different locations, at the same or different times,under control of the same entity or different entities sharing abusiness relationship.

V. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Production of T Cell-Derived PSCs (TiPSCs)

For the production of iPS cells, T cells were isolated from a bloodsample and activated prior to retroviral reprogramming to iPSCs. First,peripheral blood mononuclear cells (PBMCs) were expanded in freshlyprepared AIM-V Medium+pen/strep/glutamine (AIV-V/ps/s/g media)(Invitrogen) plus 300 IU/ml rhIL2 (Peprotech) and 10 ng/ml solubleanti-CD3 antibody (OKT3 clone, eBiosciences) and anti-CD28 antibody.Several days after activation exponential growth was verified by CEDEXcell count. After 3 days in culture cells were assayed for T-cellphenotype and then transduced with the reprogramming factors.

Retroviral vectors Nanog RFP, Lin28 RFP, Oct4 eGFP, and Sox2 eGFP wereconstructed as described previously (see U.S. Application No.61/088,054, incorporated herein by reference). Retroviral vectors c-MycRFP, Klf4 RFP, Oct4 eGFP, and Sox2 eGFP were constructed similarly.

The CD3- and CD28-activated peripheral mononuclear cells were culturedin T cell medium comprising AIM-V medium containing 2% human AB serumand 10 ng/mL IL-2. At day 6, the T cells were transfected with 6reprogramming factors through electroporation using the Amaxa U-014program (1-5×10⁶ cells/transfection, Amaxa T cell transfectionsolution). Up to day 25 post-transfection, the cells were cultured onretronectin (0.3 μg/cm²)- and vitronectin (0.2 μg/cm²)-coated wells of6-well plate at one transfection per well and with gradual transitionfrom T cell to E8 PSC medium beginning on day 14.

Activated and expanding T cells displayed characteristic cell morphologyand clustering behavior. Detection of retroviral transduction efficiencywas determined by GFP and RFP expression 72 h post initial transduction,over the course of ˜3 weeks the transgenes were silenced and display anhES cell phenotype. Well defined iPS cell colonies began to appear onday 23. GFP and RFP silencing was verified by fluorescent microscopy andcolonies were picked in a dissecting hood using a pipette tip. Colonypieces were then transferred to fresh 6 well plates. The number ofcolonies were counted to estimate reprogramming efficiency given thenumber of input plated cells. From this point clonal colonies were feddaily and manually passaged one more time and then expanded to producethe TiPSCs lines.

Example 2 TiPSC Differentiation to Hematopoietic Precursor Cells (HPCs)

Various episomally and virally reprogrammed iPSCs (Table 1), includingthe TiPSCs of Example 1, were subjected to the 3D differentiationprotocol for the production of HPCs (FIG. 1). First, the iPSCs wereacclimatized to hypoxic conditions for 5-10 passages under feeder-freeconditions on Matrigel™- or Vitronectin-coated ultra-low attachment(ULA) plates in Essential 8 (E8) media. Aggregates were made from subconfluent iPSCs at a density of 0.25-0.5 million cells per ml in thepresence Serum Free Defined (SFD) media supplemented with 5 uMblebbistatin. The process was performed in ultra-low attachment (ULA)plates or spinner flasks in SFD basal medium containing 75% IMDM(Invitrogen 12200-069) (with Glutamine and 25 mM HEPES+P/S), 25% HamsF12 (Mediatech 10-080-CV), 0.5% N2-supplement (Invitrogen 17502-048), 1%B27 supplement without retinoic acid (Invitrogen 12587-010), 0.05% BSA,50 ug/ml Ascorbic acid, and 4.5×10⁻⁴ M monothioglycerol.

Once the EBs had formed, differentiation was initiated by supplementingthe SFD basal media with 50 ng/ml of BMP-4, VEGF, and FGF2 for the first4 days. On the fifth day of differentiating the EBs, the cultures wereplaced in the presence of Flt-3 Ligand, IL3, IL6, SCF, and TPO each at50 ng/ml and heparin at 5000 units. The EB cultures were supplementedwith half the volume of fresh differentiation media containing cytokinesevery 2 days during the differentiation process until day 12-16 ofdifferentiation under hypoxic conditions. The cells were harvested afterthe differentiation process and the phenotype was assessed by flowcytometry and the functional capability was assessed using the CFUassay. The cells were harvested and the percentage of CD43/CD34 cellswas quantified by flow cytometry (FIG. 1B). The efficiency of theprocess was calculated by dividing the absolute number of HPCs generatedper input number of iPS cells (FIG. 1C).

TABLE 1 Process Validation using Multiple iPSC Lines ReprogrammingSource material Cell line Method for reprogramming 01501.102 EpisomalProgenitor cells Blood Male TiPSCs1E Viral T cells Male 1.025T Viral Tcells Female 2.022B Episomal LCL Male 2.0224B Episomal LCL Female01279.107.3902 Episomal Progenitor cells blood Male 01279.107.3908Episomal Progenitor cells blood Male 01279.107.3904 Episomal Progenitorcells blood Male 01279 Episomal Progenitor cells blood Male 01629Episomal Progenitor cells blood Male

For flow cytometry analysis, the cells were collected and washed oncewith media. The cell pellet was digested using TrypLE™ or 0.5% trypsinfor 5-10 minutes in a 37° C. incubator followed by washes with media andpassaged through a 70-μm cell strainer. The cells were resuspended inPBS-FBS containing FACS buffer, counted to estimate cell viability andstained with fluorochrome-conjugated monoclonal antibodies: anti-humanCD43 (1G10), anti-human CD31 (WM-59), anti-human CD41 (HIPS); anti-humanCD45 (HI30); anti-human CD34 (581, 8G12) (BD Biosciences San Jose,Calif.); and anti-human CD235. Non-viable cells were excluded with7-aminoactinomycin D (7-AAD, BD Biosciences). Live cell analysis wasperformed on a FACSCalibur™ or Accuri flow cytometer and Cell Questsoftware.

For the clonogenic hematopoietic progenitors assay (CFU assay) the EBswere dispersed into single cell suspensions using TryplE or 0.5%trypsin/EDTA. Viable cells were quantified, plated (50,000-300,000 cellsper mL), and assayed in humidified chambers for hematopoietic CFCs inusing Human Methylcellulose Complete Media (R&D Systems, Minneapolis,Minn.) containing stem cell factor (SCF) 50 ng/mL, erythropoietin (EPO)3 U/mL, granulocyte-macrophage colony-stimulating factor (GM-CSF) 10ng/mL, interleukin-3 (IL-3) 10 ng/mL. After 14 days the colonies werescored according to their morphology and colonies per 10⁵ cells platedwere quantified.

Example 3 Modified iPSC Differentiation to Hematopoietic Precursor Cells(HPCs)

The 1C T-cell derived iPSCs (TiPSC, derived by retroviral reprogramming)were differentiated to CD34⁺ hematopoietic progenitors through aggregatesuspension (3D) culture. 1C cells were maintained under feeder-freeconditions on Matrigel™- or Vitronectin-coated 6-well plates inEssential 8 (E8) medium. Aggregates were made from sub-confluent 1Ccells (<80% confluence) at a density of 0.5-1 million cells per ml inthe Essential 3 (E3) medium (containing only 3 of 8 components of E8medium: DMEM/F12 basal medium, ascorbic acid 2-phosphate magnesium andsodium selenite) supplemented with 50 ng/ml FGF2, 50 ng/ml VEGF, 2 μMCHIR99021 (GSK-3 inhibitor), and 10 μM blebbistatin (myosin-IIinhibitor). The aggregate formation was performed during 24 hour culturein ultra-low attachment (ULA) flasks under continuous agitation on therocker platform at 15 rpm (including all subsequent culture steps).

The formed cell aggregates (i.e., embryoid bodies—EBs) were furthertransferred to serum-free differentiation medium (50% IMDM, 50% Hams F12medium, 100 μg/ml polyvinyl alcohol, 100 μg/ml recombinant human serumalbumin, lx non-essential amino acid supplement (Invitrogen), 0.1×chemically-defined lipid supplement (Invitrogen), 125 μM ascorbic acid2-phosphate magnesium, 0.25 μM linoleic acid, trace element supplementsA (0.3×), B (0.2×) and C (0.1×) (Corning), 5 mM sodium chloride, 100 μMmonothioglycerol, 20 μM ethanolamine, 100 ng/ml heparin, and 10 ng/mlIGF1) supplemented with hematopoietic mesoderm inducing cytokines—25ng/ml BMP4, 50 mg/ml VEGF and 50 ng/ml FGF2. Cultures were continued for4 days with complete medium change on the second day.

To support differentiation and expansion of hematopoietic CD34+progenitors, cell aggregates were further transferred to serum-freedifferentiation medium (as above) supplemented with hematopoieticsupportive cytokines—50 ng/ml SCF, 20 mg/ml TPO, 10 ng/ml FLT3L, 20ng/ml IL-3, and 25 ng/ml BMP4. Cultures were continued for 4 days withcomplete medium change on the second day.

The cultures were harvested during Days 7-9 of the differentiationprocess. A single cell suspension was obtained through digestion ofdifferentiated cell aggregates in the Accutase (or Accumax) solution for15-20 min at 37° C. Cells were washed in MACS buffer (e.g., PBScontaining 5 mg/ml BSA and 1 mM EDTA), filtrated through 70 μM cellstrainers and labeled with direct CD34 paramagnetic microbeads (MiltenyiBiotec) 30 min at 4° C. CD34⁺ cells were isolated using MS or LSmagnetic columns, appropriate magnets and standard separation proceduresaccording to recommendations from manufacturer (Miltenyi Biotec).Isolated CD34⁺ cells were plated to T/NK differentiation cultures orcryopreserved for later use within 1 hour after isolation.

Example 4 Lymphoid Differentiation of HPCs

To determine the parameters for lymphoid differentiation of the HPCs ofExample 2 and Example 3, the cell lines were subjected to cultureconditions for T and NK cell differentiation. First, several variableswere tested for T cell differentiation were tested in a stroma dependentprotocol. The day 12 HPCs from different cell lines were tested for Tcell potential on stromal lines, including OP9 bone marrow stromal cellsand MS5 murine bone marrow stromal cells. The cells were cultured inαMEM media with 20% FBS, 10 ng/mL SCF, 5 ng/mL Flt-3 and 5 ng/mL IL-7.The cells were refreshed by a half-medium change three times a week.Analysis of the cells for the presence of T cells showed that the cellshad a tendency to generate myeloid cells and the presence of CD3⁺ cellscould not be detected. In addition, the stromal co-cultures performedpoorly under hypoxic conditions.

TABLE 2 Choice of matrix for lymphoid differentiation. Cells plated onRetronectin-DLL4 revealed the presence of pre T cells (CD5⁺/CD7⁺) cells.Matrix for T cell Differentiation % CD5 % CD7 % CD5/CD7 Retronectin-DLL4 11%  40%  11% Tenascin-DLL4 0.7% 5.2% 0.7% Vitronectin-DLL4 0.6%   6%0.6%

TABLE 3 Hypoxia favors T cell differentiation. Cells differentiatingunder hypoxic conditions revealed the presence of T and NK cells.CD56+/CD3− Matrix for T cell Differentiation CD3 CD4 CD8 CD3/CD4 CD3/CD4CD4/CD8 (NK cells) Hypoxia 7% 11.4%  49% 2.6% 2% 5.6% 28% Normoxia 0.2  57% 4.7% 0 0 0 6%

Accordingly, a feeder free T cell differentiation protocol wasdeveloped. The HPCs were plated on non-treated tissue culture platescoated with Retronectin and Notch DLL4 at 0.5 μg/cm² at a cell densityof about 5,000 to about 25,000 cells/cm². The HPCs were cultured inStemSpan Serum-Free Expansion Medium II (SFEM; StemCell Technologies)media supplemented with 1% Glutamax, 1% Penicillin Streptomycin, 95 μMAscorbic acid (WAKO labs), as well as 50 ng/mL of IL-7, SCF, Flt-3, andTPO (Peprotech). The media was replenished every 48 hours and at 2 weeksthe cells were split to new ligand coated plates. In addition, between 2and 3 weeks the cells were analyzed for the presence of pre-T cells bythe cell surface markers CD5 and CD7. At 4 weeks, the cells wereanalyzed for the presence of T cells by the cell surface markers CD3,CD4 and CD8. At 6-8 weeks, the cells were analyzed for the presence of Tand NK cells using the cell surface markers CD4, CD8, CD3, CD94 andCD56.

One of the parameters tested for its effect on T cell differentiationwas the choice of the matrix coating on the culture plates. A comparisonwas performed by analyzing the emergence of pre-T cells under serum freeconditions using various matrix combinations with Notch DLL4 with cordblood cells at 3 weeks post-plating. The results showed that thecombination of retronectin with DLL4 was more effective atdifferentiating the cord blood cells to pre-T cells than the combinationwith vitronectin or tenascin (Table 2).

Surprisingly, it was found that hypoxic conditions enhance feeder-free Tcell differentiation. Specifically, it was observed that hypoxiaresulted in an increase in the percentage of cells positive for CD8 anda decrease in the percentage of cells positive for CD4 as compared tothe cell differentiated under normoxic conditions (Table 3).

The efficiency of differentiating the blood cell-derived iPSCs tolymphoid lineages was analyzed by harvesting the various cell lines atday 5, day 7, day 9 and day 11 of the HPC differentiation described inExample 2. The HPC cells were thawed and plated on Retronectin and DLL4coated plates. The cells were fed fresh media every 2 days and wereanalyzed for pre-T cells markers at 2 weeks, T cell markers at 4 weeksand T and NK cell markers at 6 weeks after the HPC cells were thawed.The cells were stained for the surface expression of CD7, CD8, CD56(FIG. 3A), CD45, CD7, CD5 (FIG. 3B), and CD56, CD8, CD3 (FIG. 3C) forthe presence of T, NK and NK/T cells. The TiPSCs and the episomallyreprogrammed 3908 cells were observed to have an increased lymphoidpotential at Days 7-11 (FIG. 3A).

To determine if the surface markers could be used to increase theefficiency of lymphoid differentiation, analysis of CD43/CD34, CD34,DLL4, CD31/CD144, and CD235 was performed on both the TiPSCs1E line andthe episomal 3902 line (FIG. 4). It was found that expression of DLL4and levels of CD235 decline at Day 11 of differentiation while CD34expression decreases and CD43 expression increases with the days ofdifferentiation. Since there is absence of lymphoid cells at day 11 ofdifferentiation, it implies that a certain threshold level of expressionof these markers is essential to prime cells towards lymphoiddifferentiation in the presence of DLL4.

Further analysis of lymphoid progenitors during HPC differentiation wasperformed by magnetic sorting of the surface markers CD31, CD34, CD144,CD43, CD45, CD6, CD335, Flk-1, and DLL4. Day 8 HPCs were sorted intoCD114/CD34, CD144/CD45, CD144/CD7, and CD144/CD34/CD45/CD7 positive andnegative fractions as well as an unsorted control (FIG. 5). Thesefractions were then subjected to the lymphoid differentiation processand analyzed for the presence of CD3⁺ cells at Day 16 (FIG. 6A). It wasobserved that each of the positive fractions displayed lymphoidpotential significantly increased as compared to the negative fractionsand the unsorted control. This was further supported by the increase infold enrichment of T cell generation from the positive fraction magneticsorting (FIG. 6B). The positive fractions were plated back on freshRet-DLL4 surface for an additional two weeks.

At 4 weeks of lymphoid differentiation, the Day 8 CD144⁺/CD7⁺ and theCD144+/CD45+ HPCs sustained generation of T cells in vitro as shown bythe percentage of CD3 positive cells in FIG. 7A. The CD3 cells wereCD335 positive, CD161 positive, and invariant T cell receptor (6B11)negative. Thus, the late state cultures have an emerging NK/T cellphenotype. In addition, the CD144⁺/CD7⁺ HPCs were shown to have anincreased efficiency at producing T cells as measured by a ratio ofinput of HPCs to output of T cells at Day 16. However, the cumulativeefficiency of the process at the end of 4 weeks was shown to be highestfor the CD144/CD34/Cd45/CD7 positive fraction.

Example 5 Myeloid Differentiation of HPCs

The CD34⁺ HPCs of Examples 2 and 3 were subjected to myeloiddifferentiation for the production of relatively pure populations ofhuman dendritic cells (DCs). The cells were cultured on low attachmenttissue culture plates or flasks for the entire process. The cells wereresuspended in serum free media containing 50 ng/mL Flt-3 ligand(Flt-3L), 50 ng/mL of Stem Cell Factor (SCF), 50 ng/mL of Thrombopoeitin(TPO), 50 ng/mL Interleukin-3 (IL-3), and 50 ng/mL Interleukin-6 (IL-6)at a density of 0.5-1×10⁶ cells/mL.

To begin the myeloid differentiation, the cells were seeded at a densitybetween 0.25-0.5 million cells per mL in Myeloid Progenitor Media (Table4) and expanded for about 2 weeks. The cells were monitored forviability and expression of CD34⁺/CD45⁺/CD43⁺ at days 4 and 8. The CD34⁺population was observed to decline and there was an emergence ofCD45⁺/CD43⁺/CD31⁺ population within the cultures. The phenotype of thecultures at this stage was predominantly CD43⁺/CD45⁺/CD31⁺/CD34^(Lo).

TABLE 4 Myeloid Progenitor Media Component Manufacturer Catalog No.Concentration Serum free media* GlutaMAX Gibco 35050 1% Pen/Strep Gibco15140 1% SCF Peprotech 300-07 50 ng/mL IL-6 Peprotech 200-06 50 ng/mLTPO Peprotech 300-18 50 ng/mL IL-3 Peprotech 200-06 50 ng/mL Flt-3LPeprotech 300-19 50 ng/ml *StemSpan ™ SFEM (Stem Cell Technologies, Cat.09650), Stem Pro 34 (Invitrogen, Cat. 10639-011), or Stemline II (Sigma,Cat. S0192) can be used as a serum free media.

When the cells revealed more than 50% expression ofCD43⁺/CD45⁺/CD31⁺/CD34⁻ cell surface markers, the cells were then placedin Myeloid Expansion Media (Table 5) for 8 days. The cells were fed withfresh media every other day. At the end of 8 days, the cultured cellshad an enriched population of myeloid cells revealing 80-90%CD43⁺/CD45⁺/CD31⁺/CD34⁻. At the end of this myeloid expansion phase, thecell number, viability, and purity was determined.

TABLE 5 Myeloid Expansion Media Component Manufacturer Catalog No.Concentration Serum free media* GlutaMAX Gibco 35050 1% GM-CSF Peprotech300-03 100 ng/mL *StemSpan ™ SFEM (Stem Cell Technologies, Cat. 09650),Stem Pro 34 (Invitrogen, Cat. 10639-011) or Stemline II (Sigma, Cat.S0192) can be used as a serum free media.

Finally, at the end of 16 days of culture, the cultures were placed inDendritic Cell Enrichment Media (Table 6). The cell density wasmaintained between 0.5-1 million cells per mL, and the cells were fedwith fresh media every four days without a spin step. There was almostno proliferation observed at this stage of differentiation. Instead, thecells were observed to stick to the low attachment plates and increasein size. At the end of one week, a sample was harvested and tested forthe presence of CD209⁺, CD1a⁺, HLA-DR⁺, CD11c+, CD14⁺, CD83⁺, and CD86⁺by flow cytometry (FIGS. 9-10). These markers predominantly stainmyeloid DCs and not plasmocytoid DCs (CD123⁺). The cells were maintainedin Dendritic Cell Enrichment Media and analyzed at various time pointsto quantify the yield and purity. Wright staining was performed oncytospin samples to confirm the classic morphology of dendritic cells.

TABLE 6 Dendritic Cell Enrichment Media Component Manufacturer CatalogNo. Cocentration Serum free media* GlutaMAX Gibco 35050 1% GM-CSFPeprotech 300-03 100 ng/mL Excyte Millipore 81-129-1 1% IL-4 Peprotech200-04  20 ng/mL TNFα Peprotech 300-01A  2.5 ng/mL

Example 6 Methods of PSC Differentiation and T Cell Expansion

PSC Differentiation to CD34+ Lympho Hematopoietic Progenitors: The 1CT-cell derived iPSCs (TiPSC, derived by retroviral reprogramming) weredifferentiated to CD34+ hematopoietic progenitors through aggregatesuspension (3D) culture. 1C cells were maintained under feeder-freeconditions on Matrigel™- or Vitronectin-coated 6-well plates inEssential 8 (E8) medium. Aggregates were made from sub-confluent 1Ccells (<80% confluence) at a density of 0.5-1 million cells per ml inthe Essential 3 (E3) medium (containing only 3 of 8 components of E8medium: DMEM/F12 basal medium, ascorbic acid 2-phosphate magnesium andsodium selenite) supplemented with, 50 ng/ml FGF2, 50 ng/ml VEGF, 2 μMCHIR99021 (GSK-3 inhibitor), and 10 μM blebbistatin (myosin-IIinhibitor). The aggregate formation was performed during 24 hour culturein ultra-low attachment (ULA) flasks under continuous agitation on therocker platform at 15 rpm (including all subsequent culture steps).

The formed cell aggregates (embryoid bodies—EBs) were furthertransferred to serum-free differentiation medium (50% IMDM, 50% Hams F12medium, 100 μg/ml polyvinyl alcohol, 100 μg/ml recombinant human serumalbumin, lx non-essential amino acid supplement (Invitrogen), 0.1×chemically-defined lipid supplement (Invitrogen), 125 μM ascorbic acid2-phosphate magnesium, 0.25 μM linoleic acid, trace element supplementsA (0.3×), B (0.2×) and C (0.1×) (Corning), 5 mM sodium chloride, 100 μMmonothioglycerol, 20 μM ethanolamine, 100 ng/ml heparin, and 10 ng/mlIGF1) supplemented with hematopoietic mesoderm inducing cytokines—25ng/ml BMP4, 50 mg/ml VEGF and 50 ng/ml FGF2. Cultures were continued for4 days with complete medium change on the second day.

To support differentiation and expansion of hematopoietic CD34+progenitors, cell aggregates were further transferred to serum-freedifferentiation medium (as above) supplemented with hematopoieticsupportive cytokines—50 ng/ml SCF, 20 mg/ml TPO, 10 ng/ml FLT3L, 20ng/ml IL-3, and 25 ng/ml BMP4. Cultures were continued for 4 days withcomplete medium change on the second day.

The cultures were harvested after 1+4+4 (total 9 days) differentiationprocess. Single cell suspension was obtained through digestion ofdifferentiated cell aggregates in the Accutase (or Accumax) solution for15-20 min at 37 C. Cells were washed in MACS buffer (PBS containing 5mg/ml BSA and 1 mM EDTA), filtrated through 70 μM cell strainers andlabeled with direct CD34 paramagnetic microbeads (Myltenyi Biotec) 30min at 4 C. CD34⁺ cells were isolated using MS or LS magnetic columns,appropriate magnets and standard separation procedures according torecommendations from manufacturer (Myltenyi Biotech). Isolated CD34⁺cells were plated to T/NK differentiation cultures or cryopreserved forlater use within 1 hour after isolation.

T/NK Differentiation Cultures: For T/NK differentiation, non-tissueculture treated plastic plates were coated with Notch ligand hDLL4-Fcchimeric protein and retronectin diluted in PBS (at 0.5 μg/cm² each).Before cell plating, coating solution was aspirated, plates washed oncewith cell culture basal medium (DMEM/F12 or other), and filled with 0.25ml/cm² T cell differentiation medium (TCDM) consisting of StemSpan SFEM(Stem Cell Technologies), GlumaMax ( 1/100), PenStrep ( 1/200), ascorbicacid magnesium phosphate (250 μM), nicotinamide (2 mM) and cytokinesSCF, TPO, FLT3L and IL7 (at 50 ng/ml each). Isolated PSC-derived CD34⁺cells were plated at 5000 cells/cm² density and cultured in hypoxic (5%02) CO₂ incubator for 2 weeks with addition of fresh TCDM culture volumeon day 3 and day 6, and exchanging a half culture volume every thirdfollowing day. Total differentiated cells were harvested by gentleresuspension and collection of non-adherent cells followed by detachmentof adherent cells by 10-15 min treatment with PBS-EDTA (0.5 mM).

T Cell Expansion Cultures: For T cell expansion, tissue culture plasticplates were coated with anti-CD3 mAb (clone OKT3) and retronectindiluted in PBS (at 0.5 μg/cm² each). Before cell plating, coatingsolution was aspirated, plates washed twice with cell culture basalmedium (DMEM/F12 or other), and filled with 0.2 ml/cm² T cell expansionmedium (TCEM) consisting of ImmunoCult XF medium (Stem CellTechnologies), GlumaMax ( 1/100), PenStrep ( 1/200), and cytokines IL2and IL7 (at 10 ng/ml each). IL15 and/or IL21 could also be added toimprove expansion. Cells harvested from T/NK differentiation cultureswere plated at 20000 cells/cm² density and cultured in hypoxic (5% 02)CO₂ incubator for 2 weeks with addition of fresh TCEM culture volume onday 3 and exchanging a half culture volume every third following day.Expanded T cells were harvested by gentle resuspension and collection ofnon-adherent cells.

Example 7 2D Protocol for Production of HPCs

01279.107.3902 MeCP2 knockout and TiPSCs1E cells were subjected to the2D differentiation protocol for the production of HPCs (FIG. 16). First,the iPSCs were acclimatized to hypoxic conditions for 5-10 passagesunder feeder-free conditions on Matrigel™- or Vitronectin-coated inEssential 8 (E8) media. iPSCs were individualized and plated on PureCoatAmine-coated 6-well plates (Corning Inc.) at a density of 25000/cm² inthe presence Serum Free Defined (SFD) media supplemented with 5 uMblebbistatin. The SFD basal medium contained 75% IMDM (Invitrogen12200-069) (with Glutamine and 25 mM HEPES+P/S), 25% Hams F12 (Mediatech10-080-CV), 0.5% N2-supplement (Invitrogen 17502-048), 1% B27 supplementwithout retinoic acid (Invitrogen 12587-010), 0.05% BSA, 50 ug/mlAscorbic acid, and 4.5×10-4 M monothioglycerol supplemented with 50ng/ml of BMP-4, VEGF, and bFGF.

Induction of hematopoietic differentiation was initiated on Day 1 byculturing in SFD basal medium containing 75% IMDM (Invitrogen 12200-069)(with Glutamine and 25 mM HEPES+P/S), 25% Hams F12 (Mediatech10-080-CV), 0.5% N2-supplement (Invitrogen 17502-048), 1% B27 supplementwithout retinoic acid (Invitrogen 12587-010), 0.05% BSA, 50 ug/mlAscorbic acid, and 4.5×10-4 M monothioglycerol supplemented with 50ng/ml of BMP-4, VEGF, and bFGF. On Day 2, the media was aspirated andthe cells were placed in fresh EB1 medium. (SFD basal medium containing75% IMDM (Invitrogen 12200-069) (with Glutamine and 25 mM HEPES+P/S),25% Hams F12 (Mediatech 10-080-CV), 0.5% N2-supplement (Invitrogen17502-048), 1% B27 supplement without retinoic acid (Invitrogen12587-010), 0.05% BSA, 50 ug/ml Ascorbic acid, and 4.5×10-4 Mmonothioglycerol supplemented with 50 ng/ml of BMP-4, VEGF, and bFGF)for an additional 48 hrs.

On Days 5-10, the media was aspirated and the cells were placed in EB2media for the next 48 hrs. The EB2 media comprised fresh SFD basalmedium containing 75% IMDM (Invitrogen 12200-069) (with Glutamine and 25mM HEPES+P/S), 25% Hams F12 (Mediatech 10-080-CV), 0.5% N2-supplement(Invitrogen 17502-048), 1% B27 supplement without retinoic acid(Invitrogen 12587-010), 0.05% BSA, 50 ug/ml Ascorbic acid, and 4.5×10-4M monothioglycerol supplemented with 50 ng/ml of Flt-3 Ligand, IL3, IL6,SCF, and TPO each at 50 ng/ml and 5000 U/ml of heparin. The cells wereharvested at day 7, 8, 9, 10 of differentiation using TrypLE and stainedfor the presence of HPC markers and lymphoid progenitors.

For T cell differentiation, the HPCs were plated on non-treated tissueculture plates coated with Retronectin and Notch DLL4 at 0.5 μg/cm² at acell density of about 5,000 to about 25,000 cells/cm². The HPCs werecultured in StemSpan Serum-Free Expansion Medium II (SFEM; StemCellTechnologies) media or SFD supplemented with 1% Glutamax, 1% PenicillinStreptomycin, 95 μM Ascorbic acid (WAKO labs), as well as 50 ng/mL ofIL-7, SCF, Flt-3, and TPO (Peprotech). The media was replenished every48 hours and at 2 weeks the cells were split non-enzymatically to newligand coated plates. In addition, between 2 and 3 weeks the cells wereanalyzed for the presence of pre-T cells by the cell surface markers CD5and CD7. At 4 weeks, the cells were analyzed for the presence of T cellsby the cell surface markers CD3, CD4 and CD8. At 6-8 weeks, the cellswere analyzed for the presence of T and NK cells using the cell surfacemarkers CD4, CD8, CD3, CD94 and CD56.

Example 8 Effect of MeCP2 Disruption on Lymphoid Differentiation

To determine the role of MeCP2 in the hematopoietic differentiationprocess, a MeCP2 knockout iPSC cell line was generated. The malewildtype (WT) 01279 iPSC cell line was engineered to knockout MeCP2 tocreate the MyCell® 01279.107.3902 cell line. Using TAL nuclease, aseries of stop codons were inserted prior to the methyl CpG bindingdomain (FIG. 17B) of MeCP2 by transfection of MeCP2 TALENs and the donorplasmid containing the stop codon insertion was followed by insertion ofLoxP flanked, PGKp-Puromycin-SV40 pA in the reverse orientation. The0.1279 iPSCs were transfected with MeCP2 TALENS and Donor plasmid p1553expressing wild-type EBNA1.

The cells positive for insertion were selected for with puromycinselection, and colonies were then picked and screened by integrationPCR. Of the screened colonies, 96% were positive for insertion by twoPCR screening reactions. Fourteen of the clones were expanded andscreened at passage 3, and eight of the clones were found to be negativefor the integration of the backbone plasmid. Thus, three of theremaining clones were sequenced through the insert and two were found tobe polyclonal. The one monoclonal line 0.1279.107.302 was selected andfully characterized for further studies. Additional clones were alsoobtained and characterized as correctly engineered. The amino acidalignment of MeCP2 variants 001, 002, 005 and 008 is depicted in FIG.17C. The variant 008 does not code for a MethylCpG binding domain.

The 01279.107.3902 MeCP2 knockout cells of Example 1 and WT 01279 cellswere subjected to the 3D differentiation protocol for the production ofHPCs (FIG. 17A). First, the iPSCs were acclimatized to hypoxicconditions for 5-10 passages under feeder-free conditions on Matrigel™-or Vitronectin-coated in Essential 8 (E8) media. Aggregates were madefrom sub confluent iPSCs at a density of 0.25-0.5 million cells per mlin the presence Serum Free Defined (SFD) media supplemented with 5 uMblebbistatin. The process was performed in ultra-low attachment (ULA)plates or spinner flasks in SFD basal medium containing 75% IMDM(Invitrogen 12200-069) (with Glutamine and 25 mM HEPES+P/S), 25% HamsF12 (Mediatech 10-080-CV), 0.5% N2-supplement (Invitrogen 17502-048), 1%B27 supplement without retinoic acid (Invitrogen 12587-010), 0.05% BSA,50 ug/ml Ascorbic acid, GlutaMAX, Pen/Strep and 4.5×10⁻⁴ Mmonothioglycerol.

Once the embryoid bodies (EBs) had formed, differentiation was initiatedby supplementing the SFD basal media with 50 ng/ml of BMP-4, VEGF, andbFGF for the first 4 days. On the fifth day, the EB cultures were placedin the presence of Flt-3 Ligand, IL3, IL6, SCF, heparin, and TPO each at50 ng/ml. The EB cultures were supplemented with half the volume offresh differentiation media containing cytokines every 2 days during thedifferentiation process until day 12-16 of differentiation under hypoxicconditions.

For lymphoid differentiation, the HPCs were plated on non-treated tissueculture plates coated with Retronectin and Notch DLL4 at 0.5 μg/cm² at acell density of about 5,000 to about 25,000 cells/cm². The HPCs werecultured in StemSpan Serum-Free Expansion Medium II (SFEM; StemCellTechnologies) media supplemented with 1% Glutamax, 1% PenicillinStreptomycin, 95 μM Ascorbic acid (WAKO labs), as well as 50 ng/mL ofIL-7, SCF, Flt-3, and TPO (Peprotech). The media was replenished every48 hours and at 2 weeks the cells were split non-enzymatically to newligand coated plates. In addition, between 2 and 3 weeks the cells wereanalyzed for the presence of pre-T cells by the cell surface markers CD5and CD7. At 4 weeks, the cells were analyzed for the presence of T cellsby the cell surface markers CD3, CD4 and CD8. At 6-8 weeks, the cellswere analyzed for the presence of T and NK cells using the cell surfacemarkers CD4, CD8, CD3, CD94 and CD56.

The efficiency of the MeCP2 knockout clones at differentiating tolymphoid lineages was analyzed by harvesting the 01279.107.3902,01279.107.3905, 01279.107.3906, 01279.107.3907, and 01279.107.3908clones at day 5, day 7, day 9 and day 11 of the HPC differentiation. TheHPC cells were thawed having been cryopreserved at the time pointpreviously described and plated on Retronectin and DLL4 coated plates.The cells were fed with fresh media every 2 days and were analyzed forpre-T cell markers at 2 weeks (FIG. 18A, 18B), T and NK cell markers at4 weeks after the HPC cells were thawed. In the analysis of the pre-Tcell markers, all of the cells except for the wild-type 01279.107.0904cells had the presence of pre-T cells identified as CD5⁺CD7⁺, CD7⁺CD45⁺and CD5⁺CD45⁺. The cells were stained for the surface expression ofCD45, CD7, and CD5 (FIG. 19) and CD56, CD8, and CD3 (FIG. 20), and thepresence of T, NK and NK/T cells were quantified.

Since the input number of cells was known the absolute number of T(CD3⁺/CD8⁺), NK (CD3⁻/CD56⁺) and NK/T cells (CD3⁺/CD56⁺) was determined.The efficiency of the process is calculated by the ratio of absolutenumber of a cell type (T, NK, or NK/T)/input number of total cells or bythe ratio of absolute number of a cell type/input number of HPCs. Thepercentage of T cells (CD3⁺/CD8⁺) (FIG. 17) and the percentage of NKcells (CD3⁻/CD56⁺) (FIG. 21) were quantified by flow cytometry underFSC-SSC gate and the lymphoid scatter gate. The quantity of emergingNK/T (CD3⁺/CD56⁺), (CD3⁺/CD8⁺), NK/T (CD3⁺/CD56⁺) and NK (CD3⁻/CD56⁺)cells were also determined. Further analysis showed that the expressionof CD235/CD7, CD144⁺/Dll4⁺, and Flk-1⁺/CD34⁺ declines at day 11 ofdifferentiation. Since there is an absence of lymphoid cells at day 11of differentiation, this may imply that a certain threshold level ofexpression of these markers is essential to prime cells towards lymphoiddifferentiation in the presence of DLL4.

Analysis of the T cell markers showed that the MeCP2 KO cell lines, butnot the MeCP2 WT cell line, had the potential for lymphoiddifferentiation. The Day 9 HPC progenitors from the MeCP2 WT cells hadessentially no CD3⁺CD8⁺ T cells while the other HPC progenitors testeddifferentiated to a population of CD3⁺CD8⁺ T cells. Thus, the knockoutof the methyl binding domain of MeCP2 enhanced the potential of the HPCprogenitors to produce T and NK cells.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of producing immune cells comprising: (a) obtaining induced pluripotent stem cells (iPSCs), wherein the iPSCs are reprogrammed from a population of somatic cells; (b) incubating the iPSCs in a first defined media comprising a GSK inhibitor, wherein the media is free or essentially free of BMP4, IL-3, Flt3 ligand, and GM-CSF to prepare cells for HPC differentiation; (c) culturing the cells produced in step (b) in a second defined media comprising BMP4, FGF2, and VEGF to promote mesoderm induction in a plurality of the cells; (d) culturing the cells produced in step (c) in a third defined media comprising IL-3 and Flt3 ligand, such that a plurality of the cells proliferate and differentiate into HPCs that exhibit increased expression of one or more of CD7, DLL4, CD144, and CD235 as compared to cells produced in step (c); and (e) culturing the HPCs produced in step (d) under conditions to promote immune cell differentiation, thereby producing immune cells.
 2. The method of claim 1, wherein the immune cells are lymphoid cells.
 3. The method of claim 2, wherein the lymphoid cells are T cells, B cells, and/or NK cells.
 4. The method of claim 2, wherein HPCs that express CD34 and CD43 are cultured under conditions to promote lymphoid differentiation.
 5. The method of claim 4, wherein culturing the HPCs to promote lymphoid differentiation comprises: (i) culturing HPCs in defined, feeder-free media on a surface coated with matrix and a Notch ligand; and (ii) maintaining the culture in the presence of one or more cytokines selected from the group consisting of SCF, TPO, IL-7, and Flt-3, thereby producing lymphoid cells.
 6. The method of claim 5, wherein the defined media comprises ascorbic acid and nicotinamide.
 7. The method of claim 6, wherein the ascorbic acid is present at a concentration of 50 μM to 1 mM.
 8. The method of claim 6, wherein the nicotinamide is present at a concentration of 0.1 mM to 5 mM.
 9. The method of claim 5, wherein the matrix is retronectin and the culture further comprises immobilized anti-CD3 antibody.
 10. The method of claim 5, wherein the Notch ligand is DLL4.
 11. The method of claim 5, wherein step (ii) is one to six weeks or two to four weeks.
 12. The method of claim 2, wherein more than 10% of the lymphoid cells are positive for at least two of the markers selected from the group consisting of CD8, CD7, CD45, CD5, CD4, and CD3.
 13. The method of claim 2, wherein the lymphoid cells retain the T cell receptor gene arrangement of the blood cells.
 14. The method of claim 1, wherein the first defined media comprises blebbistatin and/or Rock inhibitors.
 15. The method of claim 1, wherein the GSK3 inhibitor is CHIR99021.
 16. The method of claim 1, wherein the first defined media further comprises VEGF, and FGF2.
 17. The method of claim 1, wherein the cells are cultured as aggregates for steps (b)-(d) and the cells are individualized prior to step (e).
 18. The method of claim 1, wherein steps (b) to (d) are performed using amine culture plates.
 19. The method of claim 1, wherein the third defined media further comprises one or more of the cytokines selected from the group consisting of IL-3, IL-6, SCF, TPO, and BMP4.
 20. The method of claim 1, wherein the third defined media comprises heparin.
 21. The method of claim 1, wherein the population of somatic cells is a population of blood cells.
 22. The method of claims 21, wherein the population of blood cells comprises T cells.
 23. The method of claim 1, wherein the HPCs differentiate into at least 20 immune cells per input HPC.
 24. The method of claim 1, further comprising isolating CD34 positive cells prior to step (e) by performing magnetic-activated cell sorting (MACS).
 25. The method of claim 24, further comprising isolating cells positive for one or more of CD7, DLL4, CD144, and CD235. 